Use of Cbl as biomarker for identifying subject suitable for treatment with anti-c-Met antibody

ABSTRACT

A method of identifying a cell sample or a subject suitable for treatment with an anti-c-Met antibody or antigen binding fragment thereof that specifically binds to an epitope within a SEMA domain of a c-Met protein by determining a Cbl concentration, a Cbl mutation, and/or a mutation of a site of c-Met for interaction with Cbl in a cell sample from a subject, as well as related compositions and methods.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of Korean Patent Application No. 10-2012-0080145, filed on Jul. 23, 2012 and Korean Patent Application No. 10-2012-0102395, filed on Sep. 14, 2012, in the Korean Intellectual Property Office, the disclosures of which are herein incorporated by reference in their entirety.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

Incorporated by reference in its entirety herein is a computer-readable nucleotide/amino acid sequence listing submitted concurrently herewith and identified as follows: One 178,498 Byte ASCII (Text) file named “712415_ST25-Revised2.TXT” created on Jul. 24, 2015.

BACKGROUND

1. Field

The present disclosure relates to a biomarker for identifying a subject suitable for treatment with anti-c-Met antibodies, a method of identifying the subject including determining a level of Cbl, a mutation of Cbl, and/or a mutation of a site of c-Met for interaction with Cbl in tumor cells of the subject, and a method for inhibiting c-Met activity and/or for preventing and/or treating c-Met-related diseases, including administering an effective amount of an anti-c-Met antibody to the identified subject.

2. Description of the Related Art

c-Met is a receptor for hepatocyte growth factor (HGF), a cytokine that binds the extracellular region of the c-Met receptor tyrosine kinase to induce cell division, movement, morphogenesis, and angiogenesis of various normal cells and tumor cells. c-Met is a representative receptor tyrosine kinase existing on the surface of cells, is itself a proto-oncogene, and is sometimes involved in various mechanisms related to cancer, such as cancer development, metastasis, migration, invasion, and angiogenesis, independent from a ligand, HGF. Thus, c-Met has been recently emerging as a new target for anti-cancer therapy.

In particular, c-Met is known to be involved in induction of resistance to commonly used anti-cancer drugs, and thus is regarded as important with respect to personalized treatments. Representative anti-cancer therapeutic drugs targeting epidermal growth factor receptor EGFR (ERBB1), i.e., Eribitux or Tarceva, work by blocking the signaling related to cancer development. In addition, Herceptin, which is well known as a breast cancer therapeutic drug, targets ERBB2 (HER2) and works by blocking the transduction of signals necessary for cell proliferation. Among patients resistant to the drugs described above, the signal transduction pathway that induces cell proliferation is not blocked due to the overexpression of c-Met. Thus, c-Met has emerged as a target of interest for many pharmaceutical companies. Still, there is a need for additional anti-c-Met antibodies and related methods and compositions.

Some of the antibodies developed were found to have adverse effects. When they retain their intrinsic structures, anti-c-Met antibodies interfere with the binding of the ligand HGF to c-Met receptor, but may also act as an agonist to trigger the signaling pathway of oncogenesis by antibody-mediated dimerization of the c-Met receptor. To avoid c-Met dimerization, anti-c-Met antibodies were structurally changed from a two-armed configuration to a one-armed configuration by genetic recombination. One-armed antagonistic antibodies to c-Met were effective when used in combination with another anticancer agent, but did not exhibit significant anticancer effects when used alone.

Cbl (E3 ligase) is known to play a leading role in the degradation of c-Met. Many c-Met inhibitors initiate Cbl-mediated c-Met degradation through ubiquitination. However, c-Met inhibitors do not show the desired therapeutic effect in patients in which Cbl cannot properly interact with c-Met due to a mutation or a quantitative reduction of Cbl or due to a mutation of c-Met.

In addition, when c-Met is activated by the ligand HGF, phosphorylation at Y1003 allows the recruitment of the Cbl enzyme to c-Met. In other words, the activation of the c-Met is a prerequisite for the recruitment of the Cbl enzyme to c-Met. Accordingly, an anticancer therapy based on Cbl-mediated c-Met degradation is executed necessarily under the condition of c-Met activation which results in an adverse effect (agonism).

There is therefore a need for a novel technique by which c-Met activity may be effectively inhibited according to kind of cancer and/or a patient's genetic makeup, with a great reduction in adverse effects.

SUMMARY

Provided is a biomarker for identifying a cancer cell and/or a patient to which an anti-c-Met antibody is applicable.

Further provided is a composition and a kit for identifying a subject suitable for application of an anti-c-Met antibody, including a biomarker.

Further provided is a method of identifying a subject suitable for application of an anti-c-Met antibody, by measuring a biomarker.

Further provided is a method of inhibiting c-Met activity, including administering a therapeutically effective amount of an anti-c-Met antibody to a subject who is identified as being suitable for treatment with an anti-c-Met antibody.

Further provided is a method of preventing and/or treating a c-Met-related disease, including administering a therapeutically effective amount of an anti-c-Met antibody to a subject who is identified as being suitable for treatment with an anti-c-Met antibody.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

These and/or other aspects will become apparent and more readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:

FIG. 1A-1D shows differences in the in vitro inhibitory activity of antibodies against c-Met, wherein

FIG. 1A is a graph showing c-Met levels in NCI-H441 cells incubated for 24 hours with anti-c-Met antibodies, as percentages of the c-Met level in the control (IgG treated, 100%), as measured by ELISA,

FIG. 1B shows Western blot photographs of the phosphorylation of kinases downstream of c-Met in NCI-H441 cells (left panel) and Caki-1 cells (right panel),

FIG. 1C is a graph showing c-Met levels in MKN45 cells incubated for 24 hours with anti-c-Met antibodies as percentages of the c-Met level in the control (IgG treated, 100%), as measured by ELISA, and

FIG. 1D is a graph showing the viability of MKN45 cells incubated for 72 hours with various concentrations of IgG (∘) or the anti-c-Met antibody L3-1Y (♦) as percentages of the viability of the control (no antibody treated, 100%), as measured by a CCK-8 assay (mean±SD);

FIG. 2A-2E shows c-Met signaling and Cbl-mediated c-Met degradation, wherein

FIG. 2A shows photographs of co-immunoprecipitation with anti-c-Met antibodies,

FIG. 2B is a graph showing c-Met levels in EBC-1 and NCI-H441 cells incubated for 4 hours with anti-c-Met antibodies as percentages of the c-Met level in the control (medium), as measured by ELISA,

FIG. 2C shows photographs of Cbl protein levels in EBC-1 and NCI-H441 cells as analyzed by Western blotting,

FIG. 2D is a graph of c-Met levels in cells plotted against treatment factors including the c-Met inhibitor PHA-665752 (Selleck Chemicals) and the anti-c-Met antibody L3-1Y or 5D5, showing that L3-1Y may trigger c-Met degradation although c-Met activity is inhibited, unlike 5D5, and

FIG. 2E is a graph showing the viability of EBC-1 cells incubated for 72 hours with various concentrations of antibodies 5D5 (∘) and L3-1Y (♦) as percentages of the viability of the control (no antibody treated, 100%), as measured by a CCK-8 assay;

FIG. 3A-3G illustrates the ability of the antibody L3-1Y to induce c-Met degradation in a Cbl-independent mechanism, wherein

FIGS. 3A and 3B are photographs showing immunoblots of protein extracts from EBC-1 cells (3A) and NCI-H441 cells (3B) incubated with L3-1Y or 5D5, detected with an anti-Ub antibody,

FIG. 3C is a graph showing c-Met levels in Cbl siRNA-transformed EBC-1 cells incubated with anti-c-Met antibodies as percentages of the c-Met level in the control (IgG treated, 100%), as measured by ELISA,

FIG. 3D is a graph showing c-Met levels in EBC-1 cells incubated with anti-c-Met antibodies, and DMSO or MG132 as percentages of the c-Met level in the control (IgG treated, 100%), as measured by ELISA,

FIG. 3E is a graph showing relative viability of HS746T cells incubated with various concentrations of antibody 5D5 (∘) or L3-1Y (♦) as measured by a CCK-8 assay,

FIG. 3F is a graph showing the relative apoptosis of HS746T cells treated with various concentrations of IgG (∘), L3-1Y (♦) and 5D5 (▴) as measured by Caspase 3/7 Glo assay, and

FIG. 3G is a graph showing the relative apoptosis of EBC-1 cells treated with various concentrations of L3-1Y and 5D5 as measured by Caspase 3/7 Glo assay;

FIG. 4A-4G illustrates the mediation of anti-c-Met antibody L3-1Y-induced c-Met degradation by LRIG1, wherein

FIG. 4A is a photograph after lysates from EBC-1 cells treated with L3-1Y were immunoprecipitaed with anti-c-Met antibody-conjugated beads and subjected to immunoblotting with the anti-LRIG1 antibody, showing that the antibody L3-1Y induces the association of c-Met and LRIG1 both present at endogenous levels,

FIG. 4B is a graph showing co-immunoprecipitation with LRIG1 and c-Met in HEK-293T cells incubated for 120 min with L3-1Y or 5D5,

FIG. 4C shows 5D5- or L3-1Y-induced apoptosis in EBC-1 cells, as measured by FACS analysis,

FIG. 4D is a graph showing the relative apoptosis of EBC-1 cells treated for 72 hours with various concentrations of L3-1Y (♦) or 5D5 (∘) as measured by Caspase 3/7 Glo assay,

FIG. 4E is a graph showing relative apoptosis of LRIG1-knockdown in EBC-1 cells treated with L3-1Y (♦),

FIG. 4F is a graph showing c-Met levels in EBC-1 cells incubated with anti-c-Met antibodies, and DMSO or concanamycin as percentages of the c-Met level in the control (IgG treated, 100%), as measured by ELISA, and

FIG. 4G shows immunofluorescence images of the co-localization of L3-1Y and lysosomes in EBC-1 cells (left panel) and MKN45 (right panel);

FIG. 5A-5D illustrates that LRIG1 mediates c-Met degradation and tumor growth inhibition in Cbl mutant tumors or Cbl-negative tumors in vivo, wherein

FIGS. 5A and 5B are graphs showing migration ability of EBC-1 cells (5C) and HS746T cells (5D), and

FIGS. 5C and 5D are graphs showing the growth of tumor volumes of EBC-1 (5C) and HS746T (5D) with time (n=15);

FIG. 6A-6F illustrates therapeutic effects of the antibody L3-1Y on tumors resistant to EGFR-targeted therapy, wherein

FIG. 6A is a photograph of immunoblots obtained with respective antibodies to p-c-Met, c-Met, EGFR, Cbl, LRIG1, and GAPDH in HCC827, HCC827 ER10, and HCC827 ER15 cells,

FIG. 6B is a graph showing the relative viability of HCC827 ER15 cells incubated for 72 hours with Erlotinib and/or L3-1Y, as measured by a CTG assay.

FIG. 6C is a graph showing c-Met levels in HCC827 ER15 cells incubated for 24 hours with anti-c-Met antibodies and 100 nM Erlotinib as percentages of the c-Met level in the control (IgG treated, 100%), as measured by ELISA,

FIG. 6D is a graph showing the levels of EGFR, Cbl, and the control GAPDH in LXFE 1422, LXFA 526, and LXFA 1647 patient samples as measured by Western blot analysis,

FIG. 6E is a graph showing RT-PCR products containing the exon 14 of the c-Met gene from LXFE 1422, LXFA 526, and LXFA 1647 samples, separated on agarose gel by electrophoresis, and

FIG. 6F shows clonogenicity of LXFE 1422, LXFA 526, and LXFA 1647, all incubated with the antibody L3-1Y, in comparison with the non-treated control;

FIG. 7 is a graph showing the conversion of erlotinib-sensitive cells into erlotinib-resistant cells; and

FIG. 8 is a schematic diagram showing the hypothesis of L3-1Y-triggered, Cbl-independent c-Met-degradation.

DETAILED DESCRIPTION

When Cbl does not interact with c-Met normally, due to a low level of the Cbl enzyme or a mutation in Cbl and/or c-Met, previously known anti-c-Met antibodies do not induce c-Met degradation and, thus, are not effective for the therapy of c-Met-related diseases. The present invention provides an anti-c-Met antibody (hereinafter referred to as “specific anti-c-Met antibody” unless otherwise stated) which initiates c-Met degradation even when the Cbl enzyme does notinteract with the c-Met normally. The specific anti-c-Met antibody is highly effective for the therapy of c-Met-related diseases in a Cbl-independent manner.

Accordingly, an embodiment provides a biomarker for identifying a subject to which the specific anti-c-Met antibody is applicable (hereinafter referred to as “anti-c-Met antibody-applicable subject”). The biomarker may be selected from the group consisting of a Cbl protein, a Cbl gene, an mRNA transcribed from a Cbl gene, a site of c-Met for interaction with Cbl, and a combination thereof. The anti-c-Met antibody-applicable subject may be a cell and/or a patient from which the cell is derived. In particular, the anti-c-Met antibody-applicable subject is cancer cells and/or patients with cancer cells.

The term “Cbl”, “Cbl protein”, or “Cbl enzyme,” as used herein, refers to E3 ligase involved in cell signalling and protein ubiquitination. This protein has functions of intracellular internalization of c-Met protein positioned on tumor cell membrane and degradation thereof. The protein may be a polypeptide encoded by the nucleotide sequence of GenBank Accession Number (NM_(—)005188, NM_(—)007619, NM_(—)170662, or NM_(—)001033238) or a polypeptide having the amino acid sequence of GenBank Accession Number (NP_(—)005199, NP_(—)031645, NP_(—)733762, or NP_(—)001028410).

The term “c-Met” or “c-Met protein” refers to a receptor tyrosine kinase (RTK) which binds hepatocyte growth factor (HGF). c-Met may be a c-Met protein from any species, particularly a mammal or primate, for instance, human c-Met (e.g., NP_(—)000236), or monkey c-Met (e.g., Macaca mulatta, NP_(—)001162100), or rodents such as mouse c-Met (e.g., NP_(—)032617.2), rat c-Met (e.g., NP_(—)113705.1), and the like. The c-Met protein may include a polypeptide encoded by the nucleotide sequence identified as GenBank Accession Number NM_(—)000245, a polypeptide having the amino acid sequence identified as GenBank Accession Number NP_(—)000236 or extracellular domains thereof. The receptor tyrosine kinase c-Met participates in various mechanisms, such as cancer development, metastasis, migration of cancer cell, invasion of cancer cell, angiogenesis, and the like.

As used herein, the term “a site of c-Met for interaction with Cbl” refers to a c-Met site that recognizes and interacts with Cbl protein, thereby allowing Cbl to perform c-Met internalization and degradation. Representative sites of c-Met for interaction with Cbl may be tyrosine at amino acid position 1003 (Y1003) to which Cbl binds, or a region encoded by exon 14 of the c-Met gene in which Y1003 is positioned. The exon 14 region of the c-Met gene ranges from the 3075th to 3215th positions on the full-length nucleotide sequence of NM_(—)000245, or corresponds to a region ranging from the 964th to 1009th positions on the full-length amino acid sequence of NP_(—)000236.

Most of the existing anti-c-Met antibodies induce c-Met internalization and degradation through interaction between Cbl and c-Met. Hence, when Cbl is absent or present at a low level in cells or when Cbl or c-Met is mutated at the site responsible for interaction with Cbl (such as a binding site), the preexisting anti-c-Met antibodies cannot induce c-Met degradation, thus rendering them therapeutically ineffective.

In contrast, the specific anti-c-Met antibody is found to induce c-Met degradation independent of whether Cbl interacts with c-Met. Hence, an embodiment of the present invention provides a method of identifying a subject to which the specific anti-c-Met antibody is applicable by measuring the level of biomarker and/or determining a mutation, whereby excellent c-Met degradation may be achieved and thus c-Met-related diseases may be effectively treated even in subjects in which the preexisting antibody therapy is ineffective.

Another embodiment provides a method of identifying (selecting) a subject suitable for application of an anti-c-Met antibody, including determining a level of Cbl, a mutation of Cbl, and/or a mutation of a site of c-Met for interaction with Cbl in a cell sample.

The method of identifying a subject suitable for application of the specific anti-c-Met antibody includes determining a Cbl concentration, a Cbl mutation, and/or a mutation of a site of c-Met for interaction with Cbl in a cell sample, wherein when Cbl is present at a low level or absent in the cell sample and/or when a mutation is present on either or both of Cbl and the site of c-Met that interacts with Cbl, the cell or the patient from which the cell is derived (separated) is determined to be a subject suitable for application of the specific anti-c-Met antibody.

As used herein, the “determining” step includes measuring the factor of interest qualitatively or quantitatively, and/or evaluating the measured results.

The cell sample may be an artificially constructed cell, a cell separated from a patient of interest, a culture of the cell, a lysate of the cell, or an extract from the cell; or a protein, DNA, and/or RNA derived from the cell, the cell culture, the cell lysate, or the cell extract. The cell may be a cancer cell (tumor cell). For example, the cancer cell may be selected from the group consisting of squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous cell carcinoma of the lung, peritoneal carcinoma, dermal cancer, dermal or intraocular melanoma, rectal cancer, perianal cancer, esophageal cancer, small intestine cancer, endocrine gland cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, chronic or acute leukemia, lymphocyte lymphoma, hepatoma, stomach cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatic tumor, breast cancer, colon cancer, large intestine cancer, endometrial cancer, uterine cancer, salivary gland cancer, renal cancer, prostate cancer, vulvar cancer, thyroid cancer, and head and neck cancer.

In one embodiment, the method is carried out using a very small amount of protein, DNA, or RNA extracted from the cell. For example, the amount of protein may range from about 0.1 to about 100 μg, from about 0.5 to about 50 μg, or from about 1 to about 10 μg, for example, from about 3 to about 7 μg, and/or the amount of DNA or RNA may range from about 0.1 to about 50 μg, from about 0.3 to about 30 μg, from about 0.5 to about 5 μg, for example from about 0.8 to about 1.2 μg suffices for the execution of the method.

Examples of the patient may be mammals including primates such as humans, monkeys, etc., and rodents such as mice, rats, etc., with preference for humans. For example, the patient may suffer from a cancer selected from the group consisting of squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous cell carcinoma of the lung, peritoneal carcinoma, dermal cancer, dermal or intraocular melanoma, rectal cancer, perianal cancer, esophageal cancer, small intestine cancer, endocrine gland cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, chronic or acute leukemia, lymphocyte lymphoma, hepatoma, stomach cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatic tumor, breast cancer, colon cancer, large intestine cancer, endometrial cancer, uterine cancer, salivary gland cancer, renal cancer, prostate cancer, vulvar cancer, thyroid cancer, head and neck cancer, and the like.

Another embodiment provides a composition for the identification (diagnosis) of a subject suitable for application of the specific anti-c-Met antibody, including at least one selected from the group consisting of a Cbl detection agent for determining a Cbl concentration, an agent for detecting a Cbl mutation, and an agent for detecting a c-Met mutation.

Another embodiment provides a kit for the identification (diagnosis) of a subject suitable for application of the specific anti-c-Met antibody, including at least one selected from the group consisting of a Cbl detection agent for determining a Cbl concentration, an agent for detecting a Cbl mutation, and an agent for detecting a c-Met mutation.

The determination of Cbl concentration may be conducted by any suitable method of determining expression, such as by mRNA detection/quantification or measuring a gene copy number, or by measuring a Cbl concentration using a protein quantification means known in the relevant art, and/or evaluating the measured results. By way of example, a Cbl concentration may be determined through an enzyme reaction, a fluorescence reaction, a luminescence action, and/or a radiation reaction using a Cbl-specific antibody or aptamer. The Cbl concentration may be analyzed using a method including, but not limited to, immunochromatography, immunohistochemistry, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), enzyme immunoassay (EIA), florescence immunoassay (FIA), luminescence immunoassay (LIA), and Western blotting. The Cbl detection agent for determining a Cbl concentration may be selected from the group consisting of an anti-Cbl antibody, an aptamer specifically binding to Cbl, and a combination thereof.

When Cbl is absent or present at a low level in cancer cells, preexisting anti-c-Met antibodies cannot induce c-Met degradation, showing a poor therapy for c-Met-related diseases. In contrast, the specific anti-c-Met antibody having a Cbl-independent activity may encourage c-Met degradation and thus is highly effective for the therapy of c-Met-related diseases even at a low or no level of Cbl. Cbl is present at a “low” level in a cancer cell or tissue sample if the level of Cbl is less than typically found in a cancer cell or tissue sample of the same type. For instance, the Cbl level of a cancer cell or tissue sample may be “low” if it is less than typically found in a cancer cell of the same type that is sensitive (not resistant) to an anti-c-Met antibody (e.g., 5D5 antibody, and the like) other than the specific anti-c-Met antibody or EGFR targeted therapy. For example, the Cbl level of a cancer cell or tissue sample may be “low” if it is less than (e.g., the minimum level) found in a cancer cell of the same type on which an anti-c-Met antibody (e.g., 5D5 antibody) other than the specific anti-c-Met antibody has therapeutic effect. Because it can be difficult to quantitatively analyze protein concentrations in a cell sample, qualitative or semi-quantitative analysis may be used as an alternative. Qualitiative methods, including immunohistochemical staining, are well known in the art. For example, the level of a protein of interest may be determined by immunohistochemical staining using an antibody (e.g., antibody #2747 (Cell signaling) for Cbl, ab36707 (Abcam) for LRIG1, and the like) that binds to a protein in a sample. The staining may be scored on a scale ranging, e.g., from ‘−’ or ‘0’ to ‘+3,’ wherein a score (stain intensity) of ‘−’ or ‘0’ represents no protein expression (no signal), score of ‘+1’ represents no or a slight protein expression (corresponding to a background signal), and scores of ‘+2’ to ‘+3’ represent progressively higher levels of protein expression (a case with a signal higher than ‘+3’ is incorporated in the score of ‘+3’). In the present invention, “the absence of Cbl or the presence of Cbl at a low level” may be defined as the stain intensity of ‘−’ or ‘+1,’ respectively, when analyzed by immunohistochemical staining using an anti-Cbl antibody or as equivalent results when analyzed by other protein analysis methods. Moreover, “the presence of LRIG1” may be defined as a stain intensity of ‘+2’ or ‘+3’ when analyzed by immunohistochemical staining using an anti-LRIG1 antibody or as equivalent results when analyzed by other protein analysis methods.

Thus, when the level of Cbl in a cell sample is determined to be ‘+1’ or ‘−’ in terms of stain intensity as measured by immunohistochemical staining using an anti-Cbl antibody, the patient from which the cell sample is originated (separated) may be determined as a subject suitable for application of the specific anti-c-Met antibody.

In one embodiment, the method of identifying a subject suitable for application of the specific anti-c-Met antibody includes:

(1′) determining a Cbl concentration in a cell sample; and

(2′) optionally, determining the subject from which the cell sample is originated as a subject suitable for application of the specific anti-c-Met antibody when Cbl is absent or present at a low level in the cell sample.

The absence of Cbl or the presence of Cbl at a low level in the cell sample may be determined as a stain intensity of ‘−’ or ‘+1’, when analyzed by immunohistochemical staining using an anti-Cbl antibody.

The Cbl mutation refers to any mutation of the nucleotide sequence of the Cbl gene and/or of the amino acid sequence or structure of Cbl protein which causes the loss of a function responsible for interaction with c-Met (e.g., binding) and/or c-Met internalization and/or c-Met degradation. In one embodiment, the Cbl mutation may be a deletion or substitution of 51 or more consecutive nucleotide residues, for example, 51 to 200 consecutive nucleotide residues, within a range from nt. 1169 to nt. 1411 of the nucleotide sequence of GenBank Accession Number NM_(—)005188, or a deletion or substitution of 17 or more consecutive amino acid residues, for example, 17 to 100 consecutive amino acid residues within a range from a.a. 343 to a.a. 424 of the amino acid sequence of GenBank Accession Number NP_(—)005179. This mutation modifies the RING finger motif of Cbl, resulting in the loss of the E3 ligase function. That is, Cbl loses the ability to degrade other proteins due to the mutation of the nucleotides or amino acids.

The Cbl mutation leading to the incapacitation of Cbl may be determined using a method including, but not limited to, the direct analysis of nucleotide sequences or amino acid sequences, RT-PCR, and DNA sequencing.

The agent for detecting a Cbl mutation may be selected from the group consisting of a probe or primer capable of detecting the mutation, an anti-Cbl antibody specifically binding to a mutated Cbl, and an aptamer specifically binding to a mutated Cbl. The probe capable of detecting the Cbl mutation may be an about 10- to about 50-mer or about 20- to about 50-mer nucleotide sequence including a mutation region of the mutated Cbl gene and/or a complementary sequence thereto, or a sequence having a similarity of about 80% or higher, about 90% or higher, or about 95% or higher therewith. The primer capable of detecting the Cbl mutation may be an about 10- to about 50-mer or about 20- to about 30-mer oligonucleotide having a nucleotide sequence capable of hybridization with 5′ and/or 3′ terminus of a mutation region (about 50 to about 200 bp including the mutation site of Cbl) of the mutated Cbl gene, wherein the nucleotide sequence capable of hybridization may be a complementary sequence thereto, or a sequence having a similarity of about 80% or higher, about 90% or higher, or about 95% or higher therewith.

In the cells wherein the interaction between Cbl and c-Met and/or c-Met internalization and/or c-Met degradation is lost, preexisting anti-c-Met antibodies cannot induce c-Met degradation and thus are unable to treat c-Met-related diseases. However, the specific anti-c-Met antibody of the present invention may encourage c-Met degradation and thus is effective for the therapy of c-Met-related diseases even upon the loss of Cbl functions.

Therefore, if the Cbl protein or a Cbl gene encoding it is found to have such a mutation in a cell sample taken from a patient, the patient may be determined to be a subject suitable for application of the specific anti-c-Met antibody.

In one embodiment, the method of identifying a subject suitable for application of the specific anti-c-Met antibody may include:

(1″) determining the presence of a mutation of a Cbl protein or a Cbl gene encoding the Cbl protein in a cell sample taken from a patient; and

(2″) optionally, deciding the cell or the patient to be a subject suitable for application of the specific anti-c-Met antibody when the mutation is present.

As used herein, the term “c-Met mutation” refers to a mutation on a c-Met site responsible for recognizing or binding Cbl, particularly to a mutation which incapacitates the interaction of Cbl with c-Met (e.g., binding) even though Cbl is present at a sufficient level or does not undergo such a mutation that leads to functional loss.

As used herein, the term “site of c-Met for interaction with Cbl” is intended to refer to a c-Met site which is recognized by Cbl so as to allow for c-Met internalization and degradation. Representative among the sites of c-Met for interaction with Cbl are tyrosine at amino acid position 1003 (Y1003) and a region encoded by exon 14 of the c-Met gene. The exon 14 region of c-Met gene ranges from nt. 3075 to nt. 3215 of the full-length nucleotide sequence of NM_(—)000245, or corresponds to a region stretching from a.a. 964 to a.a. 1009 of the full-length amino acid sequence of NP_(—)000236. The c-Met mutation may be a deletion or a substitution of tyrosine at position 1003 (Y1003) with another amino acid residue (e.g., alanine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, valine, asparagine, cysteine, glutamine, glycine, serine, threonine, aspartic acid, glutamine acid, arginine, histidine, and lysine, and preferably phenylalanine), or a deletion or a substitution of 141 or more consecutive nucleotide residues, for example, 141 to 300 consecutive nucleotide residues of exon 14 of the c-Met gene with other nucleotide residues, and/or a deletion or a substitution of 46 or more consecutive amino acid residues, for example, 46 to 100 consecutive amino acid residues of a polypeptide encoded by exon 14 with other amino acid residues. In an embodiment, the c-Met mutation may be a deletion of tyrosine at position 1003 of c-Met (Y1003), a substitution of the tyrosine residue with phenylalanine (Y1003F), a deletion of exon 14 of the c-Met gene, or a deletion of the polypeptide encoded by exon 14.

The c-Met mutation may be determined using a method including, but not limited to, the direct analysis of nucleotide sequences or amino acid sequences, RT-PCR, and DNA sequencing. The agent for detecting a c-Met mutation may be selected from the group consisting of a probe or primer capable of detecting the mutation as described above, an anti-Cbl antibody specifically binding to a mutated Cbl, and an aptamer specifically binding to a mutated Cbl.

Whereas preexisting anti-c-Met antibodies cannot induce Cbl-mediated c-Met degradation in the cells where a c-Met site which is recognized by Cbl or binds Cbl is mutated and thus are unable to treat c-Met-related diseases, the specific anti-c-Met antibody may successfully treat c-Met-related activity due to its Cbl-independent activity.

When the cell sample is found to have such a mutation on c-Met protein or c-Met gene encoding the protein, the cell or the patient from which the cell sample is taken (separated) may be determined to be a subject suitable for application of the specific anti-c-Met antibody.

In one embodiment, the method of identifying a subject suitable for application of the specific anti-c-Met antibody includes:

(1′″) determining the presence of a mutation on a site of c-Met for interaction with Cbl or on a region of c-Met gene corresponding to the site of c-Met for interaction with Cbl in a cell sample taken from a patient; and

(2′″) optionally, determining the cell or the patient to be a subject suitable for application of the specific anti-c-Met antibody when the mutation is present.

Unlike preexisting anti-c-Met antibodies, the specific anti-c-Met antibody of the present invention exhibits a Cbl-independent c-Met degradation activity because the antibody induces LRIG1 (leucine-rich repeats and immunoglobulin-like domains protein 1) to bind to c-Met, the resulting LRIG1-c-Met being internalized and degraded. Accordingly, the specific anti-c-Met antibody may act as an effective negative regulator of c-Met in the presence of LRIG1 in cells, particularly, when LRIG1 is overexpressed in cells.

“LRIG1 (Leucine-rich repeats and immunoglobulin-like domains protein 1)” refers to a transmembrane protein which interacts with receptor tyrosine kinases of the EGFR-family, MET, and RET. LRIG1 may be derived from mammals including primates such as humans and monkeys, and rodents such as rats and mice. For example LRIG1 may be human LRIG1 (Accession No. NM_(—)015541 or NP_(—)056356).

The determination of the presence/absence and/or an intracellular level of LRIG1 may be determined by any suitable method of determining expression, such as by mRNA detection/quantification, or by measuring an LRIG1 level in a cell sample by use of a protein quantification means and/or evaluating the measurement. By way of example, the presence/absence and/or an intracellular level of LRIG1 may be determined by measuring fluorescence, luminescence, and/or radiation intensity after reaction with an LRIGI-specific antibody or aptamer in conjunction with an enzyme. In detail, the presence/absence and/or an intracellular level of LRIG1 may be analyzed using a method including, but not limited to, immunochromatography, immunohistochemistry, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), enzyme immunoassay (EIA), florescence immunoassay (FIA), luminescence immunoassay (LIA), and Western blotting.

In an embodiment, the presence of LRIG1, particularly at a high level in cells, may be defined as the stain intensity of +2 or +3 when analyzed by immunohistochemical staining using an anti-LRIG1 antibody or as equivalent results when analyzed by other protein analysis methods.

Hence, the method of identifying a subject suitable for application of the specific anti-c-Met antibody may further include determining the level of LRIG1 in a cell sample. In detail, the method of identifying a subject suitable for application of the specific anti-c-Met antibody may further include (1-1), (1-1′), (1-1″), or (1-1′″) determining a level of LRIG1 in a cell sample ahead of the step (2), (2′), (2″), or (2′″), respectively. In addition, the method may further include determining the cell or the patient to be a subject suitable for application of the specific anti-c-Met antibody when LRIG1 is present. For example, the presence of LRIG1 in the cell sample is defined as a stain intensity of ‘+2’ or ‘+3’ when analyzed by immunohistochemical staining using an anti-LRIG1 antibody.

In detail, the method of identifying a subject suitable for application of the specific anti-c-Met antibody includes:

(1) determining a Cbl concentration, a Cbl mutation, and/or a mutation of a site of c-Met for interaction with Cbl in a cell sample taken from a patient;

(1-1) determining a level of LRIG1 in the cell sample; and

(2) optionally, determining the cell or the patient to be a subject suitable for application of the specific anti-c-Met antibody when Cbl is present at a low level or absent in the cell sample (wherein the Cbl concentration is expressed as a stain intensity of ‘−’ or ‘+1’ as analyzed by immunohistochemical staining using an anti-Cbl antibody) and/or when a mutation is present on either or both Cbl and the site of c-Met for interaction with Cbl if the LRIG1 is present (for example, the case that the level of LRIG1 in the cell sample is expressed as a stain intensity of ‘+2’ or ‘+3’ as analyzed by immunohistochemical staining using an anti-LRIG1 antibody).

In another embodiment, the method of identifying a subject suitable for application of the specific anti-c-Met antibody includes:

(1′) determining a Cbl concentration in a cell sample taken from a patient;

(1-1′) determining a level of LRIG1 in the cell sample; and

(2′) optionally, determining the patient to be a subject suitable for application of the specific anti-c-Met antibody when Cbl is absent or present at a low level in the cell sample while LRIG1 is present.

The absence of Cbl or the presence of Cbl at a low level in the cell sample may be defined as a stain intensity of ‘−’ or ‘+1’, when analyzed by immunohistochemical staining using an anti-Cbl antibody, and the presence of LRIG1 in the cell sample may be expressed as a stain intensity of ‘+2’ or ‘+3’ as analyzed by immunohistochemical staining using an anti-LRIG1 antibody.

In another embodiment, the method of identifying a subject suitable for application of the specific anti-c-Met antibody may include:

(1″) determining the presence of a mutation on a Cbl protein or a Cbl gene coding for the Cbl protein in a cell sample taken from a patient;

(1-1″) determining a level of LRIG1 in the cell sample; and

(2″) optionally, determining the cell or the patient to be a subject suitable for application of the specific anti-c-Met antibody when the mutation is present while LRIG1 is present,

wherein the presence of LRIG1 in the cell sample may be expressed as a stain intensity of ‘+2’ or ‘+3’ as analyzed by immunohistochemical staining using an anti-LRIG1 antibody.

In another embodiment, the method of identifying a subject suitable for application of the specific anti-c-Met antibody includes:

(1′″) determining the presence of a mutation on a site of c-Met for interaction with Cbl or on a region of c-Met gene corresponding to the site of c-Met for interaction with Cbl in a cell sample taken from a patient;

(1-1′″) determining a level of LRIG1 in the cell sample; and

(2′″) optionally, determining the cell or the patient to be a subject suitable for application of the specific anti-c-Met antibody when the mutation is present while LRIG1 is present,

wherein the presence of LRIG1 in the cell sample may be expressed as a stain intensity of ‘+2’ or ‘+3’ as analyzed by immunohistochemical staining using an anti-LRIG1 antibody.

Contemplated according to another embodiment of the present invention is a composition for the identification of a subject suitable for application of the specific anti-c-Met antibody including at least one selected from the group consisting of a Cbl detection agent for determining a Cbl concentration, an agent for detecting a Cbl mutation and an agent for detecting a c-Met mutation, and an LRIG1 detection agent for determining an LRIG1 level.

In another embodiment, the kit for the identification of a subject suitable for application of the specific anti-c-Met antibody may further include an LRIG1 detection agent for determining an LRIG1 level. In detail, the kit includes:

at least one selected from the group consisting of a Cbl detection agent for determining a Cbl concentration, an agent for detecting a Cbl mutation, and an agent for detecting a c-Met mutation;

an LRIG1 detection agent for determining an LRIG1 level; and

a detection means.

The LRIG1 detection agent for determining an LRIG1 level may be selected from the group consisting of anti-LRIG1 antibody, an aptamer specifically binding to LRIG1, and a combination thereof.

The detection means used in the kit may be any means conventionally used in determining a Cbl concentration, a Cbl mutation, a c-Met mutation, and/or an LRIG1 level. A person having ordinary skill in the art to which the present invention pertains may readily take a suitable detection means.

In accordance with still another aspect thereof, the present invention addresses a method for inhibiting c-Met activity, including administering a pharmaceutically effective amount of the specific anti-c-Met antibody to the identified subject.

In accordance with yet a further aspect thereof, the present invention addresses a method for the prophylaxis and/or therapy of c-Met-related diseases, including administering a pharmaceutically effective amount of the specific anti-c-Met antibody to a subject in need thereof.

The methods for inhibiting c-Met activity or for preventing and/or treating of c-Met-related diseases may further include identifying a subject suitable for application of the specific anti-c-Met antibody.

This identifying step is the same as in the identifying method described above. Thus, it may be carried out in the same manner as in the identifying method.

In one embodiment, the method for inhibiting c-Met activity or for the prophylaxis or therapy of c-Met-related diseases includes:

identifying a subject suitable for application of the specific anti-c-Met antibody; and

administering a pharmaceutically effective amount of the specific anti-c-Met antibody to the subject.

In another embodiment, the method for inhibiting c-Met activity or for the prophylaxis or therapy of c-Met-related diseases includes:

(1) determining a Cbl concentration, a Cbl mutation, and/or a mutation of a site of c-Met for interaction with Cbl in a cell sample taken from a patient;

(2) determining the cell or the patient to be a subject suitable for application of the specific anti-c-Met antibody when Cbl is present at a low level or absent in the cell sample and/or when a mutation is present on either or both Cbl and the site of c-Met for interaction with Cbl; and

(3) administering an effective amount of the specific anti-c-Met antibody to the cell or the patient,

wherein the absence of Cbl or the presence of Cbl at a low level in the cell sample is defined as a stain intensity of ‘−’ or ‘+1’, when analyzed by immunohistochemical staining using an anti-Cbl antibody.

In another embodiment, the method for inhibiting c-Met activity or for the prophylaxis or therapy of c-Met-related diseases includes:

(1′) determining a Cbl concentration in a cell sample taken from a patient;

(2′) determining the cell or the patient to be a subject suitable for application of the specific anti-c-Met antibody when Cbl is absent or present at a low level in the cell sample; and

(3) administering an effective amount of the specific anti-c-Met antibody to the cell or the patient,

wherein the absence of Cbl or the presence of Cbl at a low level in the cell sample is defined as a stain intensity of ‘−’ or ‘+1’, when analyzed by immunohistochemical staining using an anti-Cbl antibody.

In another embodiment, the method for inhibiting c-Met activity or for the prophylaxis and/or therapy of c-Met-related diseases includes:

(1″) determining the presence of a mutation on a Cbl protein or a Cbl gene coding for the Cbl protein in a cell sample taken from a patient;

(2″) determining the cell or the patient to be a subject suitable for application of the specific anti-c-Met antibody when the mutation is present; and

(3) administering an effective amount of the specific anti-c-Met antibody to the cell or the patient.

In another embodiment, the method for inhibiting c-Met activity or for the prophylaxis and/or therapy of c-Met-related diseases includes:

(1′″) determining the presence of a mutation on a site of c-Met for interaction with Cbl or on a region of a c-Met gene corresponding to the site of c-Met for interaction with Cbl in a cell sample taken from a patient; and

(2′″) determining the cell or the patient to be a subject suitable for application of the specific anti-c-Met antibody; and

(3) administering an effective amount of the specific anti-c-Met antibody to the cell or the patient.

As described above, the method for the prophylaxis and/or therapy of c-Met-related diseases may further include (1-1), (1-1′), (1-1″), or (1-1′″) determining a level of LRIG1 in a cell sample ahead of the step (2), (2′), (2″), or (2′″), respectively.

The specific anti-c-Met antibody of the present invention is applied to a subject. Thus, the application of the specific anti-c-Met antibody of the present invention to the subject forms an aspect of the present invention. In detail, the present invention provides the use of the specific anti-c-Met antibody in application or administration to a cell or a patient in which the absence of Cbl or the presence of Cbl at a low level (the Cbl concentration is expressed as a stain intensity of ‘−’ or ‘+1’ as analyzed by immunohistochemical staining using an anti-Cbl antibody) and/or in which a functional mutation is present on a Cbl protein or a Cbl gene and/or in which a mutation is present on a site of c-Met for interaction with Cbl or on a region of a c-Met gene corresponding to the site of c-Met for interaction with Cbl, thereby inhibiting c-Met activity or treating a c-Met-related disease. The c-Met-related disease is as described above.

In one embodiment, the present invention provides the use of the specific anti-c-Met antibody in application or administration to a cell or a patient in which the absence of Cbl or the presence of Cbl at a low level (the Cbl concentration is expressed as a stain intensity of ‘−’ or ‘+1’ as analyzed by immunohistochemical staining using an anti-Cbl antibody) and/or in which a functional mutation is present on a Cbl protein or a Cbl gene and/or in which a mutation is present on a site of c-Met for interaction with Cbl or on a region of c-Met gene corresponding to the site of c-Met for interaction with Cbl, with the proviso that LRIG1 is present in the cell sample (for example, wherein the level of LRIG1 in the cell sample is expressed as a stain intensity of ‘+2’ or ‘+3’ as analyzed by immunohistochemical staining using an anti-LRIG1 antibody), thereby inhibiting c-Met activity or treating a c-Met-related disease.

In another embodiment, the present invention provides the use of the specific anti-c-Met antibody in preparing a drug applicable to a cell in which the absence of Cbl or the presence of Cbl at a low level (the Cbl concentration is expressed as a stain intensity of ‘−’ or ‘+1’ as analyzed by immunohistochemical staining using an anti-Cbl antibody) and/or in which a functional mutation is present on a Cbl protein or a Cbl gene and/or in which a mutation is present on a site of c-Met for interaction with Cbl or on a region of c-Met gene corresponding to the site of c-Met for interaction with Cbl, thereby inhibiting c-Met activity or treating a c-Met-related disease. The drug may be a c-Met inhibitor or a therapeutic for a c-Met-related disease.

In another embodiment, the present invention provides the use of the specific anti-c-Met antibody in preparing a c-Met inhibitor and/or a therapeutic for a c-Met-related disease, applicable or administrable to a cell or a patient in which the absence of Cbl or the presence of Cbl at a low level (the Cbl concentration is expressed as a stain intensity of ‘−’ or ‘+1’ as analyzed by immunohistochemical staining using an anti-Cbl antibody) and/or in which a functional mutation is present on a Cbl protein or a Cbl gene and/or in which a mutation is present on a site of c-Met for interaction with Cbl or on a region of a c-Met gene corresponding to the site of c-Met for interaction with Cbl, with the proviso that LRIG1 is present in the cell sample (wherein, for example, the level of LRIG1 in the cell sample is expressed as a stain intensity of ‘+2’ or ‘+3’ as analyzed by immunohistochemical staining using an anti-LRIG1 antibody), thereby inhibiting c-Met activity or treating a c-Met-related disease.

The term “c-Met-related disease” refers to any disease caused by the expression or overexpression of c-Met. Cancer is representative of a c-Met-related disease. Examples of cancer include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous cell carcinoma of the lung, peritoneal carcinoma, dermal cancer, dermal or intraocular melanoma, rectal cancer, perianal cancer, esophageal cancer, small intestine cancer, endocrine gland cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, chronic or acute leukemia, lymphocyte lymphoma, hepatoma, stomach cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatic tumor, breast cancer, colon cancer, large intestine cancer, endometrial cancer, uterine cancer, salivary gland cancer, renal cancer, prostate cancer, vulvar cancer, thyroid cancer, and head and neck cancer, but are not limited thereto. Gestational diabetes also falls within the scope of c-Met-related diseases.

The term “pharmaceutically effective amount” or “therapeutically effective amount” means a dosage of a particular active agent, in this case the specific anti-c-Met antibody, to exhibit a desired effect, for instance inhibiting (degrading) c-Met, and preventing or treating c-Met-related diseases in a subject in need thereof, and may vary depending on various factors including a desired result, kinds of diseases or symptoms, the severity of illness, the route of administration, dosage forms, etc.

As described above, “c-Met” or “c-Met protein” is receptor tyrosine kinase (RTK) which binds hepatocyte growth factor (HGF). c-Met may be a c-Met protein from any species, particularly a mammal, for instance, primates such as human c-Met (e.g., NP_(—)000236), or monkey c-Met (e.g., Macaca mulatta, NP_(—)001162100), or rodents such as mouse c-Met (e.g., NP_(—)032617.2), rat c-Met (e.g., NP_(—)113705.1), and the like. The c-Met protein may include a polypeptide encoded by the nucleotide sequence identified as GenBank Accession Number NM_(—)000245, a polypeptide having the amino acid sequence identified as GenBank Accession Number NP_(—)000236 or extracellular domains thereof. The receptor tyrosine kinase c-Met participates in various mechanisms, such as cancer development, metastasis, migration of cancer cell, invasion of cancer cell, angiogenesis, and the like.

The antigen binding fragment of the anti-c-Met antibody may refer to a fragment including an antigen binding region of the anti-c-Met antibody, and be selected from the group consisting of a complementarity determining region (CDR), fragment including CDR and Fc region, scFv, (scFv)2, Fab, Fab′, and F(ab′)2 of the anti-c-Met antibody.

The anti-c-Met antibody may also include a variant of the antibody. The variant of the antibody may be any isotypes of antibodies derived from human and other animals and/or one including any Fc region of antibodies (e.g., IgA, IgD, IgE, IgG (IgG1, IgG2, IgG3, or IgG4), IgM, and the like) derived from human and other animals, having mutated hinge wherein at least one amino acid is changed, deleted or added. Unless stated otherwise, the anti-c-Met antibody may include variants of the antibody as well as the antibody with no variation.

Unless stated otherwise, the term “specific anti-c-Met antibody,” as used herein, is intended to mean an antibody or an antigen-binding fragment. The specific anti-c-Met antibody may recognize a specific region of c-Met, e.g., a specific region of the SEMA domain, as an epitope. It may be any antibody or an antigen-binding fragment that acts on c-Met to induce c-Met intracellular internalization and degradation.

c-Met, a receptor for hepatocyte growth factor, may be divided into three portions: extracellular, transmembrane, and intracellular. The extracellular portion is composed of an α-subunit and a β-subunit which are linked to each other through a disulfide bond, and contains a SEMA domain responsible for binding HGF, a PSI domain (plexin-semaphorins-integrin homology domain) and an IPT domain (immunoglobulin-like fold shared by plexins and transcriptional factors domain). The SEMA domain of c-Met protein may have the amino acid sequence of SEQ ID NO: 79, and is an extracellular domain that functions to bind HGF. A specific region of the SEMA domain, that is, a region having the amino acid sequence of SEQ ID NO: 71, which corresponds to a range from a.a. 106 to a.a. 124, is a loop region between the second and the third propellers within the epitopes of the SEMA domain. It acts as an epitope for the specific anti-c-Met antibody of the present invention.

The term “epitope,” as used herein, refers to an antigenic determinant, a part of an antigen recognized by an antibody. In one embodiment, the epitope may be a region including 5 or more consecutive amino acid residues within the SEMA domain (SEQ ID NO: 79) of c-Met protein, for instance, 5 to 19 consecutive amino acid residues corresponding to a range from a.a. 106 to a.a. 124 within the SEMA domain (SEQ ID NO: 79) of a c-Met protein. For example, the epitope may be a polypeptide having 5 to 19 consecutive amino acids selected from among partial combinations of the amino acid sequence of SEQ ID NO: 71, with the amino sequence of SEQ ID NO: 73 (EEPSQ) serving as an essential element for the epitope. For example, the epitope may be a polypeptide including the amino acid sequence of SEQ ID NO: 71, SEQ ID NO: 72, or SEQ ID NO: 73. As used herein, the term “consecutive amino acid residues” refers to amino acid residues positioned consecutively in an amino acid sequence or three-dimensional structure.

The epitope having the amino acid sequence of SEQ ID NO: 72 corresponds to the outermost part of the loop between the second and third propellers within the SEMA domain of a c-Met protein, the epitope having the amino acid sequence of SEQ ID NO: 73 is a site to which the antibody or an antigen-binding fragment according to one embodiment of the present invention most specifically binds.

Thus, the specific anti-c-Met antibody may specifically bind to an epitope which has 5 to 19 consecutive amino acids, selected from among partial combinations of the amino acid sequence of SEQ ID NO: 71, including SEQ ID NO: 73 as an essential element. For example, the anti c-Met antibody may specifically bind to an epitope including the amino acid sequence of SEQ ID NO: 71, SEQ ID NO: 72, or SEQ ID NO: 73.

In one embodiment, the specific anti-c-Met antibody may be an antibody or an antigen-binding fragment which includes:

a heavy chain variable region including the amino acid sequence of at least one heavy chain complementarity determining region (CDR) selected from the group consisting of CDR-H1 including the amino acid sequence of SEQ ID NO: 4; CDR-H2 including the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 2, or including an amino acid sequence of 8 to 19 consecutive amino acids including amino acid residues from 3rd to 10th positions within the amino acid sequence of SEQ ID NO: 2; and CDR-H3 including the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 85, or including an amino acid sequence of 6 to 13 consecutive amino acids including amino acid residues from 1st to 6th positions within the amino acid sequence of SEQ ID NO: 85; and

a light chain variable region including the amino acid sequence of at least one light chain complementarity determining region (CDR) selected from the group consisting of CDR-L1 including the amino acid sequence of SEQ ID NO: 7, CDR-L2 including the amino acid sequence of SEQ ID NO: 8, and CDR-L3 including the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 86, or including an amino acid sequence of 9 to 17 consecutive amino acids including amino acid residues from 1st to 9th positions within the amino acid sequence of SEQ ID NO: 89.

Herein, the amino acid sequences of SEQ ID NOS: 4 to 9 are respectively represented by following Formulas I to VI, below:

Formula I (SEQ ID NO: 4) Xaa₁-Xaa₂-Tyr-Tyr-Met-Ser,

wherein Xaa₁ is absent or Pro or Ser, and Xaa₂ is Glu or Asp,

Formula II (SEQ ID NO: 5) Arg-Asn-Xaa₃-Xaa₄-Asn-Gly-Xaa₅-Thr,

wherein Xaa₃ is Asn or Lys, Xaa₄ is Ala or Val, and Xaa₅ is Asn or Thr,

Formula III (SEQ ID NO: 6) Asp-Asn-Trp-Leu-Xaa₆-Tyr,

wherein Xaa₆ is Ser or Thr,

Formula IV (SEQ ID NO: 7) Lys-Ser-Ser-Xaa₇-Ser-Leu-Leu-Ala-Xaa₈-Gly-Asn- Xaa₉-Xaa₁₀-Asn-Tyr-Leu-Ala

wherein Xaa₇ is His, Arg, Gln, or Lys, Xaa₈ is Ser or Trp, Xaa₉ is His or Gln, and Xaa₁₀ is Lys or Asn,

Formula V (SEQ ID NO: 8) Trp-Xaa₁₁-Ser-Xaa₁₂-Arg-Val-Xaa₁₃

wherein Xaa₁₁ is Ala or Gly, Xaa₁₂ is Thr or Lys, and Xaa₁₃ is Ser or Pro, and

Formula VI (SEQ ID NO: 9) Xaa₁₄-Gln-Ser-Tyr-Ser-Xaa₁₅-Pro-Xaa₁₆-Thr

wherein Xaa₁₄ is Gly, Ala, or Gln, Xaa₁₅ is Arg, His, Ser, Ala, Gly, or Lys, and Xaa₁₆ is Leu, Tyr, Phe, or Met.

In one embodiment, the CDR-H1 may have an amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 22, 23, and 24. The CDR-H2 may have an amino acid sequence selected from the group consisting of SEQ ID NOS: 2, 25, and 26. The CDR-H3 may have an amino acid sequence selected from the group consisting of SEQ ID NOS: 3, 27, 28, and 85.

The CDR-L1 may have an amino acid sequence selected from the group consisting of SEQ ID NOS: 10, 29, 30, 31, 32, 33, and 108. The CDR-L2 may have an amino acid sequence selected from the group consisting of SEQ ID NOS: 11, 34, 35, and 36. The CDR-L3 may have an amino acid sequence selected from the group consisting of SEQ ID NOS: 12, 13, 14, 15, 16, 37, 86, and 89.

In another embodiment, the antibody or an antigen-binding fragment of the present invention includes a heavy variable region including a polypeptide (CDR-H1) having an amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 22, 23, and 24, a polypeptide (CDR-H2) having an amino acid sequence selected from the group consisting of SEQ ID NOS: 2, 25, and 26, and a polypeptide (CDR-H3) having an amino acid sequence selected from the group consisting of SEQ ID NOS: 3, 27, 28, and 85; and a light variable region including a polypeptide (CDR-L1) having an amino acid sequence selected from the group consisting of SEQ ID NOS: 10, 29, 30, 31, 32, 33 and 108, a polypeptide (CDR-L2) having an amino acid sequence selected from the group consisting of SEQ ID NOS: 11, 34, 35, and 36, and a polypeptide (CDR-L3) having an amino acid sequence selected from the group consisting of SEQ ID NOS 12, 13, 14, 15, 16, 37, 86, and 89.

Animal-derived antibodies produced by immunizing non-immune animals with a desired antigen generally invoke immunogenicity when injected to humans for the purpose of medical treatment, and thus chimeric antibodies have been developed to inhibit such immunogenicity. Chimeric antibodies are prepared by replacing constant regions of animal-derived antibodies that cause an anti-isotype response with constant regions of human antibodies by genetic engineering. Chimeric antibodies are considerably improved in an anti-isotype response compared to animal-derived antibodies, but animal-derived amino acids still have variable regions, so that chimeric antibodies have side effects with respect to a potential anti-idiotype response. Humanized antibodies are developed to reduce such side effects. Humanized antibodies are produced by grafting complementarity determining regions (CDR) which serve an important role in antigen binding in variable regions of chimeric antibodies into a human antibody framework.

The most important thing in CDR grafting to produce humanized antibodies is choosing the optimized human antibodies for accepting CDR of animal-derived antibodies. Antibody databases, analysis of a crystal structure, and technology for molecule modeling are used. However, even when the CDRs of animal-derived antibodies are grafted to the most optimized human antibody framework, amino acids positioned in a framework of the animal-derived CDRs affecting antigen binding are present. Therefore, in many cases, antigen binding affinity is not maintained, and thus application of additional antibody engineering technology for recovering the antigen binding affinity is necessary.

The anti-c-Met antibodies may be mouse-derived antibodies, mouse-human chimeric antibodies, humanized antibodies, or human antibodies. The antibodies or antigen-binding fragments thereof may be isolated from a living body.

The antibody may be a monoclonal antibody. The monoclonal antibody may be produced from a hybridoma of Accession No. KCLRF-BP-00220 deposited on Oct. 6, 2009, at the Korean Cell Line Research Foundation located at the Cancer Research Institute, Seoul National University College of Medicine, 28 Yongon-gong, Chongno-Gu, Seoul, 110-744, Korea.

An intact antibody includes two full-length light chains and two full-length heavy chains, in which each light chain is linked to a heavy chain by disulfide bonds. The antibody has a heavy chain constant region and a light chain constant region. The heavy chain constant region is of a gamma (γ), mu (μ), alpha (α), delta (δ), or epsilon (ε) type, which may be further categorized as gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1), or alpha 2 (α2). The light chain constant region is of either a kappa (κ) or lambda (λ) type.

As used herein, the term “heavy chain” refers to full-length heavy chain, and fragments thereof, including a variable region VH that includes amino acid sequences sufficient to provide specificity to antigens, and three constant regions, CH1, CH2, and CH3, and a hinge. The term “light chain” refers to a full-length light chain and fragments thereof, including a variable region VL that includes amino acid sequences sufficient to provide specificity to antigens, and a constant region CL.

The term “complementarity determining region (CDR)” refers to an amino acid sequence found in a hyper variable region of a heavy chain or a light chain of immunoglobulin. The heavy and light chains may respectively include three CDRs (CDRH1, CDRH2, and CDRH3; and CDRL1, CDRL2, and CDRL3). The CDR may provide contact residues that play an important role in the binding of antibodies to antigens or epitopes. The terms “specifically binding” or “specifically recognized” is well known to one of ordinary skill in the art, and indicates that an antibody and an antigen specifically interact with each other to lead to an immunological activity.

In one embodiment, the antibody may be an antigen-binding fragment selected from the group consisting of scFv, (scFv)2, Fab, Fab′, and F(ab′)2.

The term “antigen-binding fragment” used herein refers to fragments of an intact immunoglobulin including portions of a polypeptide including antigen-binding regions having the ability to specifically bind to the antigen. For example, the antigen-binding fragment may be scFv, (scFv)2, Fab, Fab′, or F(ab′)2, but is not limited thereto. Among the antigen-binding fragments, Fab that includes light chain and heavy chain variable regions, a light chain constant region, and a first heavy chain constant region CH1, has one antigen-binding site.

The Fab′ fragment is different from the Fab fragment, in that Fab′ includes a hinge region with at least one cysteine residue at the C-terminal of CH1.

The F(ab′)2 antibody is formed through disulfide bridging of the cysteine residues in the hinge region of the Fab′ fragment. Fv is the smallest antibody fragment with only a heavy chain variable region and a light chain variable region. Recombination techniques of generating the Fv fragment are widely known in the art.

Two-chain Fv includes a heavy chain variable region and a light chain region which are linked by a non-covalent bond. Single-chain Fv generally includes a heavy chain variable region and a light chain variable region which are linked by a covalent bond via a peptide linker or linked at the C-terminals to have a dimer structure like the two-chain Fv. The antigen-binding fragments may be attainable using protease (for example, the Fab fragment may be obtained by restricted cleavage of a whole antibody with papain, and the F(ab′)2 fragment may be obtained by cleavage with pepsin), or may be prepared by using a genetic recombination technique.

The term “hinge region,” as used herein, refers to a region between CH1 and CH2 domains within the heavy chain of an antibody which functions to provide flexibility for the antigen-binding site.

When an animal antibody undergoes a chimerization process, the IgG1 hinge of animal origin is replaced with a human IgG1 hinge while the disulfide bridges between two heavy chains are reduced from three to two in number. In addition, an animal-derived IgG1 hinge is shorter than a human IgG1 hinge. Accordingly, the rigidity of the hinge is changed. Thus, a modification of the hinge region may bring about an improvement in the antigen binding efficiency of the humanized antibody. The modification of the hinge region through amino acid deletion, addition, or substitution is well-known to those skilled in the art.

In one embodiment, the specific anti-c-Met antibody or an antigen-binding fragment thereof may be modified by the deletion, addition, or substitution of at least one amino acid residue on the amino acid sequence of the hinge region so that it exhibits enhanced antigen-binding efficiency. For example, the antibody may include a hinge region having the amino acid sequence of SEQ ID NO: 100, 101, 102, 103, 104, or 105. Preferably, the hinge region has the amino acid sequence of SEQ ID NO: 100 or 101.

In one embodiment of the specific anti-c-Met antibody or an antigen-binding fragment, the variable domain of the heavy chain includes the amino acid sequence of SEQ ID NO: 17, 74, 87, 90, 91, 92, 93, or 94 and the variable domain of the light chain includes the amino acid sequence of SEQ ID NO: 18, 19, 20, 21, 75, 88, 95, 96, 97, 98, 99 or 109.

In one embodiment, the specific anti-c-Met antibody is a monoclonal antibody, produced by the hybridoma cell line deposited with Accession No. KCLRF-BP-00220, binding specifically to the extracellular region of c-Met protein (refer to Korean Patent Publication No. 2011-0017698, the disclosure of which is incorporated in its entirety herein by reference).

The specific anti-c-Met antibody may include all the antibodies defined in Korean Patent Publication No. 2011-0017698, which is incorporated herein by reference.

By way of further example, the anti-c-Met antibody or antibody fragment may include a heavy chain including the amino acid sequence of SEQ ID NO: 62 (wherein the amino acid sequence from 1st to 17th positions is a signal peptide) or the amino acid sequence from 18th to 462nd positions of SEQ ID NO: 62 and a light chain including the amino acid sequence of SEQ ID NO: 68 (wherein the amino acid sequence from 1st to 20th positions is a signal peptide) or the amino acid sequence from 21st to 240th positions of SEQ ID NO: 68; or a heavy chain including the amino acid sequence of SEQ ID NO: 64 (wherein the amino acid sequence from 1st to 17th positions is a signal peptide) or the amino acid sequence from 18th to 461st positions of SEQ ID NO: 64 and a light chain including the amino acid sequence of SEQ ID NO: 68 or the amino acid sequence from 21st to 240th of SEQ ID NO: 68; or a heavy chain including the amino acid sequence of SEQ ID NO: 66 (wherein the amino acid sequence from 1st to 17th positions is a signal peptide) or the amino acid sequence from 18th to 460th positions of SEQ ID NO: 66 and a light chain including the amino acid sequence of SEQ ID NO: 68 or the amino acid sequence from 21st to 240th of SEQ ID NO: 68.

Additional examples of anti-c-Met antibodies include those in which the anti-c-Met antibody includes a heavy chain including the amino acid sequence of SEQ ID NO: 62 or the amino acid sequence from 18th to 462nd positions of SEQ ID NO: 62 and a light chain including the amino acid sequence of SEQ ID NO: 70 (wherein the amino acid sequence from 1st to 20th positions is a signal peptide) or the amino acid sequence from 21st to 240th positions of SEQ ID NO: 70; a heavy chain including the amino acid sequence of SEQ ID NO: 64 or the amino acid sequence from 18th to 461st positions of SEQ ID NO: 64 and a light chain including the amino acid sequence of SEQ ID NO: 70 or the amino acid sequence from 21st to 240th positions of SEQ ID NO: 70; or a heavy chain including the amino acid sequence of SEQ ID NO: 66 or the amino acid sequence from 18th to 460th of SEQ ID NO: 66 and a light chain including the amino acid sequence of SEQ ID NO: 70 or the amino acid sequence from 21st to 240th positions of SEQ ID NO: 70.

In still other examples, the anti-c-Met antibody may include a heavy chain including the amino acid sequence of SEQ ID NO: 62 or the amino acid sequence from 18th to 462nd positions of SEQ ID NO: 62 and a light chain including the amino acid sequence of SEQ ID NO: 110; a heavy chain including the amino acid sequence of SEQ ID NO: 64 or the amino acid sequence from 18th to 461st positions of SEQ ID NO: 64 and a light chain including the amino acid sequence of SEQ ID NO: 110; or a heavy chain including the amino acid sequence of SEQ ID NO: 66 or the amino acid sequence from 18th to 460th positions of SEQ ID NO: 66 and a light chain including the amino acid sequence of SEQ ID NO: 110.

In an embodiment, the anti-c-Met antibody may include a heavy chain including the amino acid sequence from 18th to 460th positions of SEQ ID NO: 66 and a light chain including the amino acid sequence from 21st to 240th positions of SEQ ID NO: 68; or a heavy chain including the amino acid sequence from 18th to 460th positions of SEQ ID NO: 66 and a light chain including the amino acid sequence of SEQ ID NO: 110.

The specific anti-c-Met or the antigen-binding fragment thereof according to the present invention may be used in a pharmaceutical composition. Accordingly, a pharmaceutical composition including a pharmaceutically effective amount of the specific anti-c-Met or the antigen-binding fragment thereof, optionally together with a pharmaceutically acceptable vehicle, a diluent, and/or an excipient, form yet another aspect of the present invention.

So long as it is usually used in drug formulations, any pharmaceutically acceptable vehicle may be contained in the pharmaceutical composition including the anti-c-Met antibody according to the present invention. Examples of the pharmaceutically acceptable vehicle available for the pharmaceutical composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinyl pyrrolidone, cellulose, water, syrup, methyl cellulose, methyl hydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil, but are not limited thereto. The pharmaceutical composition may further include an additive selected from the group consisting of a diluent, an excipient, a lubricant, a wetting agent, a sweetener, a flavorant, an emulsifier, a suspending agent, a preservative, and a combination thereof.

The specific anti-c-Met or the pharmaceutical composition including a pharmaceutically effective amount thereof may be administered orally or parenterally. For parenteral administration, the administration may be carried out via intravenous, subcutaneous, intramuscular, intraperitoneal, intradermal, local, intranasal, intrapulmonary, and intrarectal routes, but is not limited thereto. For oral administration, however, the pharmaceutical composition is preferably coated or formulated to protect the active ingredient from being degraded in the stomach because the peptides are digested by pepsin. In addition, the administration may be performed with the aid of an instrument adapted for delivering the pharmaceutical composition to target cells.

The effective amount may vary depending on various factors including the type of formulation, the patient's age, weight, and sex, the severity of the disorder being treated, diet, the time of administration, the route of administration, the rate of excretion, and sensitivity. For example, the composition of the present invention may be administered at a single dose raging from about 0.001 mg to about 100 mg for adults, for example, from about 0.001 mg to about 0.1 mg, from about 0.1 mg to about 1 mg, from about 1 mg to about 10 mg, or from about 10 mg to about 100 mg.

According to a method that is well known to those skilled in the art, the anti-c-Met antibody or the pharmaceutical composition of the present invention may be formulated, together with pharmaceutically acceptable carriers and/or excipients, into unit dose forms, or may be included within a multiple dose package. In this context, the pharmaceutical composition may be formulated into solutions in oil or aqueous media, suspensions, syrup, emulsions, elixirs, powders, granules, tablets, or capsules, and may further include a dispersant or a stabilizer.

The anti-c-Met antibody or the pharmaceutical composition of the present invention may be administered alone or in combination with other therapeutics. In this case, they are administered sequentially or simultaneously together with conventional therapeutics.

The composition including an antibody or an antigen-binding fragment can be formulated into immunoliposomes. Liposomes including an antibody may be prepared using methods that are well-known in the art. The immunoliposomes may be produced from a lipid composition including phosphatidylcholine, cholesterol, and PEGylated phosphatidylethanolamine by reverse-phase evaporation. To quote an example, Fab′ may be conjugated to liposomes by disulfide reformation. The liposome may further contain an anticancer agent such as doxorubicin.

In one embodiment, the antibody may act as an antagonist of c-Met protein.

As used herein, the term “antagonist” is intended to encompass all molecules that at least partially block, suppress, or neutralize at least one of the biological activities of a target (e.g., c-Met). By way of example, an “antagonist” antibody means an antibody that represents suppression or inhibition against the biological activity of the antigen to which the antibody binds (e.g., c-Met). An antagonist may function to reduce ligand-induced receptor phosphorylation or to incapacitate or kill cells which have been activated by ligands. Also, an antagonist may completely interfere with receptor-ligand interaction or substantially reduce the interaction by changing the three-dimensional structure of the receptor or by down regulation.

The present invention may expand the choice of application of the specific anti-c-Met antibody. That is, the present invention may expand the choice of application of an anti-c-Met antibody by identifying the cells (cancer cells) on which preexisting anti-c-Met antibodies cannot induce c-Met degradation due to a quantitative reduction in Cbl or a mutation on Cbl or c-Met, or patients with the cells, thereby effectively treating c-Met-related diseases through a Cbl-independent c-Met degradation mechanism. Accordingly, the present invention enables a tailored treatment pertinent to patients of c-Met-related diseases.

Further, anticancer agents based on Cbl-mediated mechanisms may exhibit the adverse effect of c-Met activation, whereas the Cbl-independent mechanism, as suggested in the present invention, allows for targeting c-Met, whether activated or not, thus reducing the adverse effects attributable to the agonism.

In addition, the present invention may identify a subject suitable for application of the specific anti-c-Met antibody by use of a very small amount of proteins or RNA, thus enjoying advantages in terms of the cost, time, and efficacy of anticancer therapy.

The present invention pertains to a specific anti-c-Met antibody which may be applied to subjects showing c-Met degradation independent of or partially dependent on Cbl. Conventional therapeutics, which induce c-Met degradation in a Cbl-dependent manner, cannot work at all when c-Met is abnormally up-regulated due to a problem with Cbl. However, the specific anti-c-Met antibody of the present invention may be used to treat c-Met-related diseases even upon the up-regulation of c-Met due to Cbl mutation. Accordingly, it is necessary to molecularly diagnose a patient population which does not allow Cbl to bind to c-Met or to function normally in order to apply the specific anti-c-Met antibody thereto.

Due to its ability to induce Cbl-independent c-Met degradation, the specific anti-c-Met antibody may be applied to a variety of cancers irrespective of the presence or absence or mutation of Cbl. Accordingly, provided herein is a method of treating cancer characterized by low or absent Cbl expression or by a mutation in Cbl or c-Met that inhibits interaction between Cbl and c-Met, or a cancer resistant to treatment with EGFR-targeted therapy in a subject, the method comprising administering an antibody or antigen-binding fragment thereof that specifically binds to an epitope within a SEMA domain of a c-Met protein to the subject. The cancer may be further characterized by the the presence of LRIG1. The method may, therefore, also include steps of (i) determining that expression of Cbl in a cell or sample or the cancer from the subject is low, or detecting a mutation in Cbl or c-Met that inhibits interaction between Cbl and c-Met, and/or (ii) detecting or otherwise confirming the presence of LRIG1 in a cell or sample of the cancer from the subject. All other aspects of this method are as described with respect to other methods provided herein.

Because preexisting anti-c-Met antibodies induce c-Met degradation only in a Cbl enzyme (E3 ligase)-dependent manner, they cannot exert anticancer activity in the case of abnormal Cbl. An experiment showed that the lung cancer cell line EBC-1 expressed a very low level of Cbl, compared to H4441, which is a different lung cancer cell line (refer to FIG. 2C). EBC-1 cells were, thus, observed to abnormally increase in c-Met protein level because of the lack of Cbl-mediated c-Met degradation. As demonstrated by an experiment (refer to FIG. 2E), the specific anti-c-Met antibody, which is able to target c-Met in a Cbl-independent manner, inhibited the growth of the lung cancer cell line EBC-1 in a dose-dependent manner, whereas the preexisting antibody 5D5 promoted the cell growth. In addition, the stomach cancer cell line HS746T in which c-Met is truncated at a site for interaction with Cbl was observed to die upon treatment with the specific anti-c-Met antibody, but proliferated in the presence of 5D5 9 (refer to FIG. 3E).

When cancer cells are further proliferated as Cbl does not work in c-Met degradation, cancer progression continues if the cell death program does not work. In order to demonstrate that the anticancer effect of the specific anti-c-Met antibody is due to its ability to down-regulate c-Met irrespective of the presence or absence of Cbl, an apoptosis experiment was carried out. The specific anti-c-Met antibody of the present invention was found to induce the lung cancer cell line EBC-1 to undergo apoptosis in a dose-dependent manner, whereas the preexisting anti-c-Met antibody 5D5 cannot at all (refer to FIG. 3G).

The anticancer effect of the specific anti-c-Met antibody irrespective of Cbl was also demonstrated in the condition where Cbl was down-regulated with siRNA or was functionally inhibited by an inhibitor.

Transfection with siRNA causes the cancer cell line to decrease in intracellular protein as well as RNA level of Cbl. Thus, if the anticancer effect of an anti-c-Met antibody is monitored in cancer cells transfected with or without siRNA of Cbl, the dependency of the antibody on Cbl may be obtained. The anticancer effect of the specific anti-c-Met antibody was almost not changed in light of c-Met degradation whereas 5D5, dependent on Cbl, did not reduce the level of c-Met in Cbl-knockdown animals (refer to FIG. 3C).

MG132 is an inhibitor which reduces the degradation of ubiquitin-conjugated proteins through the proteasome pathway. E3 ligases including Cbl function to degrade proteins only through the proteasome pathway. Even after treatment with MG132, the specific anti-c-Met antibody was found to decrease the level of c-Met in cancer cells (refer to FIG. 3D), which demonstrates the independency of the specific anti-c-Met antibody on Cbl.

In addition, the adverse effects caused by preexisting antibodies were not detected with the specific anti-c-Met antibody of the present invention. 5D5, a preexisting anti-c-Met antibody, induces the activation of c-Met (phosphorylation at C-terminus Y1234 or Y1235). Thus, the phosphorylation of Akt and Erk oncogenes downstream of c-Met activation takes place, causing the adverse effect of the drug (agonism). Phosphorylated Akt and phosphorylated Erk are known as markers for determining agonism. In the lung cancer cell line NCI-H441 and the kidney cancer cell line Caki-1, adverse effects were significantly detected after the application of 5D5 to the cells, but little or not at all after the application of the specific anti-c-Met antibody, as measured for the agonism marker (refer to FIG. 1B). This data indicates that an anti-c-Met antibody which targets c-Met in a Cbl-independent manner exhibits improved anticancer effects with a reduction in side effects.

Further, a mouse tumor xenograft experiment and a cell migration assay demonstrated the independency of the specific anti-c-Met antibody of the present invention on Cbl in stomach and lung cancer cell lines (refer to Example 5, below). The anticancer effect of the specific anti-c-Met antibody was observed in vivo using a tumor xenograft experiment in which nude mice were subcutaneously transplanted with cancer cells and treated with drugs. In tumor xenograft experiments with mice transplanted with the lung cancer cell line EBC-1, which is low in intracellular Cbl level, or the stomach cancer cell line HS746T, which has a mutation on the site of c-Met for interaction with Cbl, 5D5 did not suppress cancer progression without a difference from the control. However, the specific anti-c-Met antibody was found to have an effective anticancer mechanism in vivo thanks to its independency on Cbl, as measured by the tumor xenograft experiment (refer to FIGS. 5C and 5D).

With the advance of cancer progression, cancer cells acquire the ability to migrate along blood vessels, which results in cancer metastasis. A cell migration assay is an experiment by which the ability of cells to migrate may be observed in vitro. The specific anti-c-Met antibody was found to suppress the migration of cancer cells, but the preexisting antibody 5D5 did not, as measured by a cell migration assay using the lung cancer cell line EBC-1 and the stomach cancer cell line HS746T in both of which Cbl independency may be proven (refer to FIGS. 5A and 5B).

One or more embodiments of the present invention will now be described in further detail with reference to the following Examples. However, these examples are for the illustrative purposes only and are not intended to limit the scope of the invention.

Reference Example 1 Construction of Anti-c-Met Antibody

1.1. Production of “AbF46”, a Mouse Antibody to c-Met

1.1.1. Immunization of Mouse

To obtain immunized mice necessary for the development of a hybridoma cell line, each of five BALB/c mice (Japan SLC, Inc.), 4 to 6 weeks old, was intraperitoneally injected with a mixture of 100 μg of human c-Met/Fc fusion protein (R&D Systems) and one volume of complete Freund's adjuvant. Two weeks after the injection, second intraperitoneal injection was conducted on the same mice with a mixture of 50 μg of human c-Met/Fc protein and one volume of incomplete Freund's adjuvant. One week after the second immunization, the immune response was finally boosted. Three days later, blood was taken from the tail and the sera were 1/1000 diluted in PBS and used to examine a titer of antibody to c-Met by ELISA. Mice found to have a sufficient antibody titer were selected for use in the cell fusion process.

1.1.2. Cell Fusion and Production of Hybridoma

Three days before cell fusion, BALB/c mice (Japan SLC, Inc.) were immunized with an intraperitoneal injection of a mixture of 50 μg of human c-Met/Fc fusion protein and one volume of PBS. The immunized mice were anesthetized before excising the spleen from the left half of the body. The spleen was meshed to separate splenocytes which were then suspended in a culture medium (DMEM, GIBCO, Invitrogen). The cell suspension was centrifuged to recover the cell layer. The splenocytes thus obtained (1×10⁸ cells) were mixed with myeloma cells (Sp2/0) (1×10⁸ cells), followed by spinning to give a cell pellet. The cell pellet was slowly suspended, treated with 45% polyethylene glycol (PEG) (1 mL) in DMEM for 1 min at 37° C., and supplemented with 1 mL of DMEM. To the cells was added 10 mL of DMEM over 10 min, after which incubation was conducted in water at 37° C. for 5 min. Then the cell volume was adjusted to 50 mL before centrifugation. The cell pellet thus formed was resuspended at a density of 1˜2×10⁵ cells/mL in a selection medium (HAT medium) and 0.1 mL of the cell suspension was allocated to each well of 96-well plates which were then incubated at 37° C. in a CO₂ incubator to establish a hybridoma cell population.

1.1.3. Selection of Hybridoma Cells Producing Monoclonal Antibodies to c-Met Protein

From the hybridoma cell population established in Reference Example 1.1.2, hybridoma cells which showed a specific response to c-Met protein were screened by ELISA using human c-Met/Fc fusion protein and human Fc protein as antigens.

Human c-Met/Fc fusion protein was seeded in an amount of 50 μL (2 μg/mL)/well to microtiter plates and allowed to adhere to the surface of each well. The antibody that remained unbound was removed by washing. For use in selecting the antibodies that do not bind c-Met but recognize Fc, human Fc protein was attached to the plate surface in the same manner.

The hybridoma cell culture obtained in Reference Example 1.1.2 was added in an amount of 50 μL to each well of the plates and incubated for 1 hour. The cells remaining unreacted were washed out with a sufficient amount of Tris-buffered saline and Tween 20 (TBST). Goat anti-mouse IgG-horseradish peroxidase (HRP) was added to the plates and incubated for 1 hour at room temperature. The plates were washed with a sufficient amount of TBST, followed by reacting the peroxidase with a substrate (OPD). Absorbance at 450 nm was measured on an ELISA reader.

Like this, hybridoma cell lines which secrete antibodies that specifically and strongly bind to human c-Met but not human Fc were selected repeatedly. From the hybridoma cell lines obtained by repeated selection, a single clone producing a monoclonal antibody was finally separated by limiting dilution. The single clone of the hybridoma cell line producing the monoclonal antibody was deposited with the Korean Cell Line Research Foundation, an international depository authority located at 28 Yongon-gong, Chongno-Gu, Seoul, Korea, on Oct. 6, 2009, with Accession No. KCLRF-BP-00220 according to the Budapest Treaty (refer to Korean Patent Laid-Open Publication No. 2011-0047698).

1.1.4. Production and Purification of Monoclonal Antibody

The hybridoma cell line obtained in Reference Example 1.1.3 was cultured in a serum-free medium, and the monoclonal antibody were produced and purified from the cell culture.

First, the hybridoma cells cultured in 50 mL of a medium (DMEM) supplemented with 10% (v/v) FBS were centrifuged and the cell pellet was washed twice or more with 20 mL of PBS to remove the FBS therefrom. Then, the cells were resuspended in 50 mL of DMEM and incubated for 3 days at 37° C. in a CO2 incubator.

After the cells were removed by centrifugation, the supernatant was stored at 4° C. before use or immediately used for the separation and purification of the antibody. An AKTA system (GE Healthcare) equipped with an affinity column (Protein G agarose column; Pharmacia, USA) was used to purify the antibody from 50 to 300 mL of the supernatant, followed by concentration with an filter (Amicon). The antibody in PBS was stored before use in the following examples.

1.2. Construction of chAbF46, a Chimeric Antibody to c-Met

A mouse antibody is apt to elicit immunogenicity in humans. To solve this problem, chAbF46, a chimeric antibody, was constructed from the mice antibody AbF46 produced in Example 1 by replacing the constant region, but not the variable region responsible for antibody specificity, with an amino sequence of human IgG1 antibody.

In this regard, a gene was designed to include the nucleotide sequence of “EcoRI-signal sequence-VH-NheI-CH-TGA-XhoI” (SEQ ID NO: 38) for a heavy chain and the nucleotide sequence of “EcoRI-signal sequence-VL-BsiWI-CL-TGA-XhoI” (SEQ ID NO: 39) for a light chain and synthesized. Then, a DNA fragment having the heavy chain nucleotide sequence (SEQ ID NO: 38) and a DNA fragment having the light chain nucleotide sequence (SEQ ID NO: 39) were digested with EcoRI (NEB, R0101S) and XhoI (NEB, R0146S) before cloning into a pOptiVECTM-TOPO TA Cloning Kit enclosed in an OptiCHOTM Antibody Express Kit (Cat no. 12762-019, Invitrogen), and a pcDNATM3.3-TOPO TA Cloning Kit (Cat no. 8300-01), respectively.

Each of the vectors thus constructed was amplified with the aid of a Qiagen Maxiprep kit (Cat no. 12662). The vectors which respectively carried the heavy chain and the light chain were co-transfected at a ratio of 4:1 (80 μg:20 μg) into 293T cells (2.5×107). The transfection into 293T cells (2.5×107) was performed in the presence of 360 μL of 2M CaCl2.

Afterwards, the cells were incubated in DMEM supplemented with 10% (v/v) FBS for 5 hours at 37° C. under a 5% CO2 condition and then in FBS-free DMEM for 48 hours at 37° C. under a 5% CO2 condition.

After centrifugation, the supernatant was applied to AKTA prime (GE Healthcare) to purify the antibody. In this regard, 100 mL of the supernatant was loaded at a flow rate of 5 mL/min to AKTA Prime equipped with a Protein A column (GE healthcare, 17-0405-03), followed by elution with an IgG elution buffer (Thermo Scientific, 21004). The buffer was exchanged with PBS to purify a chimeric antibody AbF46 (hereinafter referred to as “chAbF46”).

1.3. Construction of Humanized Antibody huAbF46 from Chimeric Antibody chAbF46

1.3.1. Heavy Chain Humanization

To design two domains H1-heavy and H3-heavy, human germline genes which share the highest homology with the VH gene of the mouse antibody AbF46 purified in Reference Example 1.2 were analyzed. An Ig BLAST result revealed that VH3-71 has a homology of 83% at the amino acid level. CDR-H1, CDR-H2, and CDR-H3 of the mouse antibody AbF46 were defined according to Kabat numbering. A design was made to introduce the CDR of the mouse antibody AbF46 into the framework of VH3-71. Hereupon, back mutations to the amino acid sequence of the mouse AbF46 were conducted at positions 30 (S→T), 48 (V→L), 73 (D→N), and 78 (T→L). Then, H1 was further mutated at positions 83 (R→K) and 84 (A→T) to finally establish H1-heavy (SEQ ID NO: 40) and H3-heavy (SEQ ID NO: 41).

For use in designing H4-heavy, human antibody frameworks were analyzed by a search for BLAST. The result revealed that the VH3 subtype, known to be most stable, is very similar in framework and sequence to the mouse antibody AbF46. CDR-H1, CDR-H2, and CDR-H3 of the mouse antibody AbF46 were defined according to Kabat numbering and introduced into the VH3 subtype to construct H4-heavy (SEQ ID NO: 42).

1.3.2. Light Chain Humanization

To design two domains H1-light (SEQ ID NO: 43) and H2-light (SEQ ID NO: 44), human germline genes which share the highest homology with the VH gene of the mouse antibody AbF46 were analyzed. An Ig BLAST search result revealed that VK4-1 has a homology of 75% at the amino acid level. CDR-L1, CDR-L2, and CDR-L3 of the mouse antibody AbF46 were defined according to Kabat numbering. A design was made to introduce the CDR of the mouse antibody AbF46 into the framework of VK4-1. Hereupon, back mutations to the amino acid sequence of the mouse AbF46 were conducted at positions 36 (Y→H), 46 (L→M), and 49 (Y→I). Only one back mutation was conducted at position 49 (Y→I) on H2-light.

To design H3-light (SEQ ID NO: 45), human germline genes which share the highest homology with the VL gene of the mouse antibody AbF46 were analyzed by a search for BLAST. As a result, VK2-40 was selected as well. VL and VK2-40 of the mouse antibody AbF46 were found to have a homology of 61% at an amino acid level. CDR-L1, CDR-L2, and CDR-L3 of the mouse antibody were defined according to Kabat numbering and introduced into the framework of VK4-1. Back mutations were conducted at positions 36 (Y→H), 46 (L→M), and 49 (Y→I) on H3-light.

For use in designing H4-light (SEQ ID NO: 46), human antibody frameworks were analyzed. A blast search revealed that the Vk1 subtype, known to be the most stable, is very similar in framework and sequence to the mouse antibody AbF46. CDR-L1, CDR-L2, and CDR-L3 of the mouse antibody AbF46 were defined according to Kabat numbering and introduced into the Vk1 subtype. Hereupon, back mutations were conducted at positions 36 (Y→H), 46 (L→M), and 49 (Y→I) on H4-light.

Thereafter, DNA fragments having the heavy chain nucleotide sequences (H1-heavy; SEQ ID NO: 47, H3-heavy; SEQ ID NO: 48, H4-heavy; SEQ ID NO: 49) and DNA fragments having the light chain nucleotide sequences (H1-light; SEQ ID NO: 50, H2-light; SEQ ID NO: 51, H3-light; SEQ ID NO: 52, H4-light; SEQ ID NO: 53) were digested with EcoRI (NEB, R0101S) and XhoI (NEB, R0146S) before cloning into a pOptiVECTM-TOPO TA Cloning Kit enclosed in an OptiCHOTM Antibody Express Kit (Cat no. 12762-019, Invitrogen) and a pcDNATM3.3-TOPO TA Cloning Kit (Cat no. 8300-01), respectively, so as to construct recombinant vectors for expressing a humanized antibody.

Each of the recombinant vectors was amplified using Qiagen Maxiprep kit (Cat no. 12662). The vectors which respectively carried the heavy chain and the light chain were co-transfected at a ratio of 4:1 (80 μg:20 μg) into 293T cells (2.5×107). The transfection into 293T cells (2.5×107) was performed in the presence of 360 μL of 2M CaCl2. Afterwards, the cells were incubated in DMEM supplemented with 10% (v/v) FBS for 5 hours at 37° C. under a 5% CO2 condition, and then in FBS-free DMEM for 48 hours at 37° C. under a 5% CO2 condition.

After centrifugation, the supernatant were applied to AKTA prime (GE Healthcare) to purify the antibody. In this regard, 100 mL of the supernatant was loaded at a flow rate of 5 mL/min to AKTA Prime equipped with a Protein A column (GE healthcare, 17-0405-03), followed by elution with an IgG elution buffer (Thermo Scientific, 21004). The buffer was exchanged with PBS to purify a humanized antibody AbF46 (hereinafter referred to as “huAbF46”). The humanized antibody huAbF46 used in the following examples included a combination of H4-heavy (SEQ ID NO: 42) and H4-light (SEQ ID NO: 46).

1.4. Construction of scFV Library of huAbF46 Antibody

For use in constructing an scFv of the huAbF46 antibody from the heavy and light chain variable regions of the huAbF46 antibody, a gene was designed to have the structure of “VH-linker-VL” for each of the heavy and the light chain variable region, with the linker having the amino acid sequence “GLGGLGGGGSGGGGSGGSSGVGS” (SEQ ID NO: 54). A polynucleotide sequence (SEQ ID NO: 55) coding for the designed scFv of huAbF46 was synthesized in Bioneer and an expression vector for the polynucleotide had the nucleotide sequence of SEQ ID NO: 56.

After expression, the product was found to exhibit specificity to c-Met.

1.5. Construction of Library Genes for Affinity Maturation

1.5.1. Selection of Target CDR and Synthesis of Primer

The affinity maturation of huAbF46 was achieved. First, six complementary determining regions (CDRs) were defined according to Kabat numbering. The CDRs are given in Table 1, below.

TABLE 1 CDR Amino Acid Sequence CDR-H1 DYYMS (SEQ ID NO: 1) CDR-H2 FIRNKANGYTTEYSASVKG (SEQ ID NO: 2) CDR-H3 DNWFAY (SEQ ID NO: 3) CDR-L1 KSSQSLLASGNQNNYLA (SEQ ID NO: 10) CDR-L2 WASTRVS (SEQ ID NO: 11) CDR-L3 QQSYSAPLT (SEQ ID NO: 12)

For use in the introduction of random sequences into the CDRs of the antibody, primers were designed as follows. Conventionally, N codons were utilized to introduce bases at the same ratio (25% A, 25% G, 25% C, 25% T) into desired sites of mutation. In this experiment, the introduction of random bases into the CDRs of huAbF46 was conducted in such a manner that, of the three nucleotides per codon in the wild-type polynucleotide encoding each CDR, the first and second nucleotides conserved over 85% of the entire sequence while the other three nucleotides were introduced at the same percentage (each 5%) and that the same possibility was imparted to the third nucleotide (33% G, 33% C, 33% T).

1.5.2. Construction of Library of huAbF46 Antibodies and Affinity for c-Met

The construction of antibody gene libraries through the introduction of random sequences was carried out using the primers synthesized in the same manner as in Reference Example 1.5.1. Two PCR products were obtained, as shown in FIG. 2, using a polynucleotide covering the scFV of huAbF46 as a template, and were subjected to overlap extension PCR to give scFv library genes for huAbF46 antibodies in which only desired CDRs were mutated. Libraries targeting each of the six CDRs prepared from the scFV library genes were constructed.

The affinity for c-Met of each library was compared to that of the wildtype. Most libraries were lower in affinity for c-Met, compared to the wild-type. The affinity for c-Met was retained in some mutants.

1.6. Selection of Antibody with Improved Affinity from Libraries

After maturation of the affinity of the constructed libraries for c-Met, the nucleotide sequence of scFv from each clone was analyzed. The nucleotide sequences thus obtained are summarized in Table 2 and were converted into IgG forms. Four antibodies which were respectively produced from clones L3-1, L3-2, L3-3, and L3-5 were used in the subsequent experiments.

TABLE 2 Library Clone constructed CDR Sequence H11-4 CDR-H1 PEYYMS (SEQ ID NO: 22) YC151 CDR-H1 PDYYMS (SEQ ID NO: 23) YC193 CDR-H1 SDYYMS (SEQ ID NO: 24) YC244 CDR-H2 RNNANGNT (SEQ ID NO: 25) YC321 CDR-H2 RNKVNGYT (SEQ ID NO: 26) YC354 CDR-H3 DNWLSY (SEQ ID NO: 27) YC374 CDR-H3 DNWLTY (SEQ ID NO: 28) L1-1 CDR-L1 KSSHSLLASGNQNNYLA (SEQ ID NO: 29) L1-3 CDR-L1 KSSRSLLSSGNHKNYLA (SEQ ID NO: 30) L1-4 CDR-L1 KSSKSLLASGNQNNYLA (SEQ ID NO: 31) L1-12 CDR-L1 KSSRSLLASGNQNNYLA (SEQ ID NO: 32) L1-22 CDR-L1 KSSHSLLASGNQNNYLA (SEQ ID NO: 33) L2-9 CDR-L2 WASKRVS (SEQ ID NO: 34) L2-12 CDR-L2 WGSTRVS (SEQ ID NO: 35) L2-16 CDR-L2 WGSTRVP (SEQ ID NO: 36) L3-1 CDR-L3 QQSYSRPYT (SEQ ID NO: 13) L3-2 CDR-L3 GQSYSRPLT (SEQ ID NO: 14) L3-3 CDR-L3 AQSYSHPFS (SEQ ID NO: 15) L3-5 CDR-L3 QQSYSRPFT (SEQ ID NO: 16) L3-32 CDR-L3 QQSYSKPFT (SEQ ID NO: 37)

1.7. Conversion of Selected Antibodies into IgG

Respective polynucleotides coding for heavy chains of the four selected antibodies were designed to have the structure of “EcoRI-signal sequence-VH-NheI-CH-XhoI” (SEQ ID NO: 38). The heavy chains of huAbF46 antibodies were used as they were because their amino acids were not changed during affinity maturation. In the case of the hinge region, however, the U6-HC7 hinge (SEQ ID NO: 57) was employed instead of the hinge of human IgG1. Genes were also designed to have the structure of “EcoRI-signal sequence-VL-BsiWI-CL-XhoI” for the light chain. Polypeptides encoding light chain variable regions of the four antibodies which were selected after the affinity maturation were synthesized in Bioneer. Then, a DNA fragment having the heavy chain nucleotide sequence (SEQ ID NO: 38) and DNA fragments having the light chain nucleotide sequences ((DNA fragment including L3-1-derived CDR-L3: SEQ ID NO: 58, DNA fragment including L3-2-derived CDR-L3: SEQ ID NO: 59, DNA fragment including L3-3-derived CDR-L3: SEQ ID NO: 60, and DNA fragment including L3-5-derived CDR-L3: SEQ ID NO: 61)) were digested with EcoRI (NEB, R0101S) and XhoI (NEB, R0146S) before cloning into a pOptiVECTM-TOPO TA Cloning Kit enclosed in an OptiCHOTM Antibody Express Kit (Cat no. 12762-019, Invitrogen) and a pcDNATM3.3-TOPO TA Cloning Kit (Cat no. 8300-01), respectively, so as to construct recombinant vectors for expressing affinity-matured antibodies.

Each of the recombinant vectors was amplified using a Qiagen Maxiprep kit (Cat no. 12662). The vectors which respectively carried the heavy chain and the light chain were co-transfected at a ratio of 4:1 (80 μg:20 μg) into 293T cells (2.5×107). The transfection into 293T cells (2.5×107) was performed in the presence of 360 μL of 2M CaCl2. Afterwards, the cells were incubated in DMEM supplemented with 10% (v/v) FBS for 5 hours at 37° C. under a 5% CO2 condition and then in FBS-free DMEM for 48 hours at 37° C. under a 5% CO2 condition.

After centrifugation, the supernatant was applied to AKTA prime (GE Healthcare) to purify the antibody. In this regard, 100 mL of the supernatant was loaded at a flow rate of 5 mL/min to AKTA Prime equipped with a Protein A column (GE healthcare, 17-0405-03), followed by elution with an IgG elution buffer (Thermo Scientific, 21004). The buffer was exchanged with PBS to purify four affinity-matured antibodies (hereinafter referred to as “huAbF46-H4-A1(L3-1 origin), huAbF46-H4-A2 (L3-2 origin), huAbF46-H4-A3 (L3-3 origin), and huAbF46-H4-A5 (L3-5 origin),” respectively).

1.8. Construction of Constant Region- and/or Hinge Region-Substituted huAbF46-H4-A1

Among the four antibodies selected in Reference Example 1.7, huAbF46-H4-A1 was found to be the highest in affinity for c-Met and the lowest in Akt phosphorylation and c-Met degradation degree. In the antibody, the hinge region, or the constant region and the hinge region, were substituted.

The antibody huAbF46-H4-A1 (U6-HC7) was composed of a heavy chain including the heavy chain variable region of huAbF46-H4-A1, U6-HC7 hinge and the constant region of human IgG1 constant region, and a light chain including the light chain variable region of huAbF46-H4-A1 and human kappa constant region. The antibody huAbF46-H4-A1 (IgG2 hinge) was composed of a heavy chain including a heavy chain variable region, a human IgG2 hinge region, and a human IgG1 constant region, and a light chain including the light chain variable region of huAbF46-H4-A1 and a human kappa constant region. The antibody huAbF46-H4-A1 (IgG2 Fc) was composed of the heavy chain variable region of huAbF46-H4-A1, a human IgG2 hinge region, and a human IgG2 constant region, and a light chain including the light variable region of huAbF46-H4-A1 and a human kappa constant region. Hereupon, the histidine residue at position 36 on the human kappa constant region of the light chain was changed into tyrosine in all of the three antibodies to increase antibody production.

For use in constructing the three antibodies, a polynucleotide (SEQ ID NO: 63) coding for a polypeptide (SEQ ID NO: 62) composed of the heavy chain variable region of huAbF46-H4-A1, a U6-HC7 hinge region, and a human IgG1 constant region, a polynucleotide (SEQ ID NO: 65) coding for a polypeptide (SEQ ID NO: 64) composed of the heavy chain variable region of huAbF46-H4-A1, a human IgG2 hinge region, and a human IgG1 region, a polynucleotide (SEQ ID NO: 67) coding for a polypeptide (SEQ ID NO: 66) composed of the heavy chain variable region of huAbF46-H4-A1, a human IgG2 region, and a human IgG2 constant region, and a polynucleotide (SEQ ID NO: 69) coding for a polypeptide (SEQ ID NO: 68) composed of the light chain variable region of huAbF46-H4-A1, with a tyrosine residue instead of histidine at position 36, and a human kappa constant region were synthesized in Bioneer. Then, the DNA fragments having heavy chain nucleotide sequences were inserted into a pOptiVECTM-TOPO TA Cloning Kit enclosed in an OptiCHOTM Antibody Express Kit (Cat no. 12762-019, Invitrogen) while DNA fragments having light chain nucleotide sequences were inserted into a pcDNATM3.3-TOPO TA Cloning Kit (Cat no. 8300-01) so as to construct vectors for expressing the antibodies.

Each of the vectors thus constructed was amplified with the aid of a Qiagen Maxiprep kit (Cat no. 12662). The vectors which respectively carried the heavy chain and the light chain were co-transfected at a ratio of 4:1 (80 μg:20 μg) into 293T cells (2.5×107). The transfection into 293T cells (2.5×107) was performed in the presence of 360 μL of 2M CaCl2. Afterwards, the cells were incubated in DMEM supplemented with 10% (v/v) FBS for 5 hours at 37° C. under a 5% CO2 condition and then in FBS-free DMEM for 48 hours at 37° C. under a 5% CO2 condition.

After centrifugation, the supernatant was applied to AKTA prime (GE Healthcare) to purify the antibody. In this regard, 100 mL of the supernatant was loaded at a flow rate of 5 mL/min to AKTA Prime equipped with a Protein A column (GE healthcare, 17-0405-03), followed by elution with IgG elution buffer (Thermo Scientific, 21004). The buffer was exchanged with PBS to finally purify three antibodies (huAbF46-H4-A1(U6-HC7), huAbF46-H4-A1(IgG2 hinge), and huAbF46-H4-A1(IgG2 Fc)). Of them, huAbF46-H4-A1(U6-HC7) was selected as a representative anti-c-Met antibody and used in the following examples. For convenience, it was named anti-c-Met antibody L3-1 Y.

Reference Example 2 Preparation of Cell Line and Cell Culture

The human stomach cancer cell line MKN45 (JCRB0254) and the lung cancer cell line EBC-1 (JCRB0820) were purchased from the Health Science Research Resource Bank (Shinjuku, Japan), and the kidney cancer cell line Caki-1 (HTB-46), the stomach cancer cell line HS746T (HTB-135), and the human lung adenocarcinoma cell line NCI-H441 (HTB-174) were purchased from the American Type Culture Collection (ATCC, Manassas, Va.).

MKN45, EBC-1, and NCI-H441 cells were maintained in RPMI1640 (GIBCO) while HS746T and Caki-1 cells were cultured in DMEM supplemented with 10% (v/v) fetal bovine serum (FBS, GIBCO) and 1% (v/v) penicillin/streptomycin (GIBCO). Cell culturing was executed at 37° C. in a humidified atmosphere with 5% CO2. Cells were subcultured before reaching confluence and counted using a CEDEX Analyzer (Roche Diagnostics).

Example 1 Effect of Anti-c-Met Antibodies on c-Met

1.1. c-Met Degradation in NCI-H441 Cells

IgG (negative control, eBioscience), the anti-c-Met antibody L3-1Y constructed in Reference Example 1, and the anti-c-Met antibody 5D5 known as an agonist (separated and purified form ATCC Cat. # HB-11895 hybridoma cells purchased from the American Type Culture Collection (ATCC, Manassas, Va.) (positive control) were tested for c-Met degradation. This test was to examine the efficacy of an antibody by comparing relative changes in total c-Met level, on the basis of the fact that when the antibody binds thereto, c-Met on the cell membrane is internalized and degraded.

NCI-H441 cells were seeded at a density of 2×105 cells/ml, together with 5 μg/ml of each antibody, into 96-well plates and incubated for 24 hours before cell lysis with the lysis buffer Complete lysis-M (Roche, 04719956001). Total c-Met levels in the cell lysates thus obtained were measured using Human Total HGF R/c-MET ELISA KIT (R&D systems, DYC358) according to the instructions of the manufacturer.

Experiments were performed in triplicate, and mean values of three measurements are presented in FIG. 1A (mean±SD). FIG. 1A shows the ability of antibodies to induce c-Met degradation as expressed as percentages relative to the c-Met level of the IgG treatment group.

As seen in FIG. 1A, NCI-H441 cells were observed to undergo more severe c-Met degradation when treated with the anti-c-Met antibodies L3-1Y and 5D5 than with IgG. In NCIH441 cells rich in Cbl, 5D5 reduced the c-Met level to an extent similar to that of L3-1Y after treatment for 24 hours, but this did not lead to a decrease in cell growth.

1.2. Phosphorylation of c-Met and Downstream Molecule

5D5, a conventional therapeutic anti-c-Met antibody, induces the activation of c-Met (phosphorylation of C-terminal Y1234 or Y1235), which may subsequently cause an adverse effect (agonism), that is, the phosphorylation of downstream molecules Akt and Erk responsible for oncogenesis. Phospho-Akt and phosphor-Erk are known as markers for determining agonism.

To examine adverse effects attributed to the agonism, phosphorylation of kinases downstream of c-Met was measured in NCI-H441 and Caki-1 cells treated with the same antibodies as in Example 1.1.

NCI-H441 and Caki-1 cells were seeded at a density of 2×105 cells/ml into respective 96-well plates and left for 24 hours before treatment with 5 μg/mL of each of the antibodies for 30 min in the absence of serum. The phosphorylation of the downstream kinases was measured using Western blotting.

The results are shown in FIG. 1B. As may be seen in FIG. 1B, L3-1Y significantly inhibited the phosphorylation of the downstream molecules in both NCI-H441 and Caki-1 cells, compared to the other antibodies, indicating that the antibody L3-1Y effectively blocks the c-Met signaling pathway. Accordingly, little or no adverse effects are detected in the lung cancer cell line NCI-H441 and the kidney cancer cell line Caki-1 treated with L3-1Y, whereas 5D5 causes significant adverse effects. The data shows that an anti-c-Met antibody which targets c-Met in a Cbl-independent manner promises an anticancer effect without side effects.

1.3. c-Met Degradation in MKN45 Cell

Total c-Met levels in MKN45 cells treated with the antibodies were measured using ELISA in the same manner as in Example 1.1, and the results are depicted in FIG. 1C. As is apparent from the data of FIG. 1C, the antibody L3-1Y significantly reduced the intracellular level of c-Met, compared to the positive control 5D5 as well as the negative control IgG. That is, the antibody L3-1Y has potent ability to trigger c-Met degradation.

1.4. Cell Viability—MKN45 Cell

After tumor cells (MKN45) were treated with the antibodies, their viability was measured using a cell proliferation assay. In this context, the cell proliferation assay was performed using CCK-8 (Dojindo Laboratories, Gaithersburg, Md., USA) according to the instructions of the manufacturer.

MKN45 cells were seeded at a density of 1×104 cells/ml/well into 96-well plates and treated at 37° C. for 72 hours with or without various concentrations (0.0032, 0.016, 0.008, 0.4, 2, and 10 μg/ml) of IgG or L3-1Y. Subsequently, CCK-8 was aliquoted at a concentration of 10 μl/well to the plates, followed by incubation at 37° C. for an additional one hour. Absorbance at 450 nm of each well was measured on an automatic ELISA reader (Molecular Devices). Cell viability was expressed as percentages of the absorbance value of the non-treated group.

The results are shown in FIG. 1D. As seen in the graph of FIG. 1D, the viability of the tumor cells was significantly reduced upon treatment with the antibody L3-1Y (♦), compared to IgG (∘).

Example 2 Cbl-Dependent c-Met Degradation 1

2.1. Induction of Interaction Between Cbl and c-Met

NCI-H441 cells were seeded at a density of 2×10⁵ cells/ml/well into 100 mm plates, and then treated with 5 μg/ml of the anti-c-Met antibody L3-1Y or 5D5 at 37° C. for 30 min in a serum-free medium. The cells were lyzed using a protease mix tablet (Roche) in the lysis buffer Complete lysis-M (Roche, 04719956001) and incubated at 4° C. with immunoprecipitating antibody-conjugated A/G agarose beads (Pierce). These beads were washed four times with a lysis buffer. The proteins bound to the beads were eluted with a sampling buffer (Invitrogen), followed by immunoblot analysis.

The results are given in FIG. 2A. As may be seen in the blots of FIG. 2A, the interaction between Cbl and c-Met was induced by the antibody 5D5, but not by the antibody L3-1Y. Cbl, although serving as a main E3 ligase to induce the quantitative reduction of c-Met, interacts with only activated c-Met and is thus highly apt to cause the adverse effect (agonism). Compared to the antibody 5D5, which induces the interaction of Cbl with c-Met, the antibody L3-1Y is deemed little causative of the adverse effect side effects because it does not induce the interaction.

2.2. c-Met Degradation in EBC-1 and NCI-H441 Cells

EBC-1 or NCI-H441 cells were seeded at a density of 2×105 cells/ml, together with 5 μg/ml of the anti-c-Met antibody L3-1Y or 5D5, into 96-well plates and incubated for 4 hours. Then, c-Met degradation was measured using ELISA in the same manner as in Example 1.1.

The results are given in FIG. 2B. As shown in FIG. 2B, the antibody L3-1Y decreased the total c-Met level by 50% or higher in both cells while the antibody 5D5 induced c-Met degradation in NCI-H441 cells to a degree similar to what is induced by L3-1Y, but almost did not in EBC-1 cells.

To examine the cause of the result, Cbl protein levels in EBC-1 and NCI-H441 cells were measured by Western blot analysis using an Cbl antibody (cell signaling). The result is given in the upper panel of FIG. 2C. In addition, as seen in FIG. 2C, Cbl protein was at a relatively high level in NCI-H441 cells, but did not exist in EBC-1 cells. Therefore, the reason why the antibody 5D5 cannot induce c-Met degradation in EBC-1 cells is the absence of Cbl protein in EBC-1 cells. Therefore, the antibody L3-1Y is concluded to trigger Cbl-independent c-Met degradation. In other words, the antibody L3-1Y may exert excellent therapeutic effects on tumors which are not sufficiently sensitive to preexisting anti-c-Met antibodies (e.g., 5D5), such as EBC-1-related tumors, exhibiting the significance that it expands the range of cancers that may be therapeutically treated with anti-c-Met antibodies.

In addition, lung cancer tissues from lung cancer patients (n=14, non-small cell lung cancer (NSCLC) from Oncotest) were analyze for Cbl protein level by Western blot using the Cbl antibody (cell signaling) in the same manner as above. The results are given in the lower panel of FIG. 2C. As may be seen in FIG. 2C, Cbl levels differ from one patient to another, which indicates that patients, although suffering from the same cancer, are less apt to be therapeutically treated with preexisting anti-c-Met antibodies (e.g., 5D5) if their Cbl protein levels are low. Therefore, if their Cbl protein levels are known or measured, patients may be treated with pertinent antibodies in consideration of personal biological properties, so that more effective personalized therapy may be achieved.

2.3. Effect of c-Met Inhibitor on c-Met Degradation: MKN45 Cell

MKN45 cells were seeded at a density of 2×105 cells/ml, together with predetermined concentrations of the anti-c-Met antibody L3-1Y or 5D5, into 96-well plates and incubated for four hours. To each well, 1 μl of PHA-665752 (Selleck Chemical) was added. For controls, 1 μl of DMSO was used, instead of the inhibitor. c-Met degradation was analyzed using ELISA in the same manner as in Example 1.1. The results are given in FIG. 2E, and show that L3-1Y may trigger c-Met degradation irrespective of whether a c-Met is activated or not.

2.4. Cell Viability—EBC-1 Cell

EBC-1 was seeded at a density of cells/ml/well into 96-well plates and incubated for 72 hours with various concentrations of the antibody 5D5 or L3-1Y (0.0032, 0.016, 0.008, 0.4, 2, and 10 μg/ml). Cell viability was measured in the same manner as in Example 1.4.

The results are given in FIG. 2E. As shown in FIG. 2E, the cell viability was significantly decreased with the antibody L3-1Y (♦), compared to the antibody 5D5 (∘).

Example 3 Cbl-Dependent c-Met Degradation 2 (Degradation Pathway)

3.1. c-Met Degradation Mechanism of Anti-c-Met Antibody 1: Ubiquitination Assay

An experiment was made to see whether Anti-c-Met antibody-triggered c-Met degradation takes place through the lysosomal pathway or the proteasome pathway mediated by E3 ligase including Cbl. In this context, poly-ubiquitination was examined by an ubiquitination assay.

Twenty-four hours after EBC-1 or NCI-H441 cells were seeded at a density of 2×105 cell/ml into 100 mm plates, they were incubated with 5 μg/ml of the antibody L3-1Y or 5D5 at 37° C. for 30, 60, or 120 min. The cells were separated and treated with a lysis buffer Complete lysis-M (Roche, 04719956001) to afford a protein extract. Ahead of the cell separation, MG132 (C26H41N3O5, Merck) or concanamycin (Merck) was added to inhibit the proteasome pathway or the lysosome pathway, respectively, so as to increase protein levels. Together with anti-c-Met antibody-conjugated NG agarose beads (Pierce), 500 μg of the protein extract was pulled down. The endogenous ubiquitination of c-Met was examined by immunoblotting using an anti-Ub antibody (Santa Cruz).

Photographs of immunoblots are given in FIGS. 3A and 3B (3A: immunoblots of EBC-1 cells, 3B: immunoblots of NCI-H441 cells). As may be seen in FIGS. 3A and 3B, the poly-ubiquitination of c-Met was observed in both the cell lines after treatment with the antibody 5D5, but neither of the cell lines after treatment with the antibody L3-1Y. Thus, the antibody 5D5 may induce the poly-ubiquitination of c-Met. but the antibody L3-1Y cannot, which indicates that the antibody L3-1Y is involved in c-Met degradation through a non-proteasome pathway, that is, a lysosome pathway.

3.2. c-Met Degradation in Cells Transformed with Cbl siRNAs: Cbl Knockdown

Dependency of the anti-c-Met antibody-triggered c-Met degradation on Cbl was examined. For this, Cbl knockdown was achieved by transforming Cbl siRNAs into EBC-1 cells which were then treated with antibodies before measuring c-Met degradation.

Cbl siRNAs (Dharmacon)-transformed EBC-1 cells which were seeded at a density of 2×105 cells/ml into plates were incubated at 37° C. for 24 hours with 5 μg/ml of the anti-c-Met antibody L3-1Y. c-Met degradation was measured using ELISA in the same manner as in Example 1.1. For control, 5 μg/ml of IgG was used.

The results are given in FIG. 3C. sictl represents a control treated with a control siRNA commercially available from Dharmacon, in comparison with siCbl. As may be seen in FIG. 3C, the total c-Met level in the Cbl siRNA-transformed EBC-1 cells treated with the antibody L3-1Y was reduced to about 30% of that in the transformed EBC-1 cells treated with the control IgG, indicating a c-Met degradation efficiency of as high as about 70%.

3.3. c-Met Mechanism of Anti-c-Met Antibody 2—Treatment with MG132

Apart from Example 3.1, the c-Met degradation mechanism of anti-c-Met antibody was examined. In this regard, c-Met degradation was measured when the proteasome pathway was blocked by the proteasome inhibitor MG132 (C26H41N3O5, Merck).

EBC-1 cells which were seeded at a density of 2×105 cells/ml into 96-well plates were incubated at 37° C. for 4 hours with the anti-c-Met antibody L3-1Y 5 μg/ml+DMSO 1 μL, or with the antibody L3-1Y 5 μg/ml+MG132 1 μL (10 μm). c-Met degradation was measured using ELISA in the same manner as in Example 1.1. For control, IgG 5 μg/ml+DMSO 1 μL, or IgG 5 μg/ml+MG132 1 μL was used.

The results are given in FIG. 3D. As may be seen in FIG. 3D, the antibody L3-1Y reduced the c-Met level by about 70% in the cells treated without the proteasome inhibitor MG132 and by about 50% in the cells treated with MG132, indicating that L3-1Y-triggered-c-Met degradation is not significantly affected by MG132 treatment. The data demonstrates again that c-Met degradation of the antibody L3-1Y does not take the Cbl-mediated proteasome pathway, but is independent of Cbl.

3.4. Cell Viability—HS746T Cell

HS746T cells were seeded at a density of 5×104 cells/ml/well into 96-well plates and incubated with various concentrations (0.0032, 0.016, 0.008, 0.4, 2, and 10 μg/ml) of the antibody 5D5 or L3-1Y at 37° C. for 72 hours. Cells were measured on the basis of the method used in the example.

HS746T is a stomach cancer cell line in which the site of c-Met for interaction with Cbl is truncated by natural mutation in all c-Met molecules.

The results are given in FIG. 3E. As may be seen in FIG. 3E, tumor cell viability was greatly reduced upon treatment with the antibody L3-1Y (♦), compared to the antibody 5D5 (∘).

3.5. Apoptosis Induction

Anti-c-Met antibodies were assayed for ability to induce apoptosis.

Apoptotic effects of IgG (control), L3-1Y, and 5D5 on cells were measured in vitro using a Caspase 3/7 Glo® assay system on HS746T cells present at a density of 5×104 cells/ml/well in 96-well plates.

In brief, EBC-1 cells in an FBS 10%(v/v) RPMI 1640 medium and HS746T cells in an FBS 10%(v/v) DMSO medium were plated at a density of 5×103 cells/well into respective black 96-well plates (Corning Incorporated), and incubated at 37° C. for 72 hours with various dilutions (0.0032, 0.016, 0.008, 0.4, and 2 μg/ml) of IgG, L3-1Y or 5D5 in a medium supplemented with 10%(v/v) FBS and then at room temperature for 30 min with 100 μL/wells of Caspase 3/7 Glo® (Promega). Luminescence signals were recorded with an Envision 2104 Multi-label Reader (Perkin Elmer). Apoptosis rates after being normalized to CCK-8 assay values are shown in FIG. 3F.

As may be seen in FIG. 3F, the antibody L3-1Y (♦) was found to induce potent apoptosis, whereas the apoptosis induced by 5D5 (▴) was weaker than the control IgG (∘).

The apoptotic effects of the antibodies L3-1Y and 5D5 on the lung cancer cell line EBC-1 were measured in the same manner and the results are given in FIG. 3G. As may be seen in FIG. 3G, the antibody L3-1Y induced higher apoptosis in a dose-dependent manner even in the lung cancer cell line EBC-1, which expresses a low level of Cbl, compared to the preexisting anti-c-Met antibody 5D5.

Example 4 LRIG1-Mediated c-Met Degradation

4.1. Induction of Interaction Between LRIG1 and c-Met by Anti-c-Met Antibody: EBC-1 Cell

An examination was made of the ability of anti-c-Met antibodies to induce c-Met to associate with LRIG1 in EBC-1 cells. An LRIG1-c-Met associated protein was separated and purified by co-immunoprecipitation and quantitatively analyzed by immunoblotting. In this regard, cells were treated with an anti-c-Met antibody for periods of time shown in FIG. 4A and harvested. The cells were lysed in a lysis buffer Complete lysis-M (Roche, 04719956001) to afford a protein extract. Together with anti-c-Met antibody-conjugated NG agarose beads (Pierce), 500 μg of the extract was pulled down, followed by immunoblotting with an anti-LRIG1 antibody (Abcam) to detect the association of c-Met with LRIG1. The data obtained above shows that the association of c-Met with LRIG1 is induced two hours after treatment with L3-1Y.

4.2. Induction of Interaction Between LRIG1 and c-Met by Anti-c-Met Antibody: HEK293 Cell

An examination was made of the ability of anti-c-Met antibodies to induce c-Met to associate with LRIG1 in HEK293 cells. An LRIG1-c-Met associated protein was separated and purified by co-immunoprecipitation and quantitatively analyzed by immunoblotting. In this regard, cells were treated with an anti-c-Met antibody for 2 hours and harvested. The cells were lysed in a lysis buffer Complete lysis-M (Roche, 04719956001) to afford a protein extract. Together with anti-c-Met antibody-conjugated NG agarose beads (Pierce), 500 μg of the extract was pulled down, followed by immunoblotting with an anti-LRIG1 antibody (Abcam) to detect the association of c-Met with LRIG1.

The immunoblotting results are shown in FIG. 4B. As is apparent from the data of FIG. 4B, the antibody L3-1Y may induce the association of LRIG1 with c-Met whereas the antibody 5D5 cannot.

4.3. Apoptosis Induction by Anti-c-Met Antibody 1

EBC-1 cells were treated for 72 hours with 1 μg/ml of L3-1Y or 5D5 and transferred to tubes which were spun at 120 rpm at 4° C. for 3 min. The cells were stained for 15 min with 5 μl of AnnexinV (BD Pharmingen) and 2 μl of PI (Propidium Iodide, 50 g/μl) under a dark condition, followed by FACS analysis (FACS CAntoll flow cytometer, Becton, Dickinson and Company). The results are shown in FIG. 4C. The annexin V(+)/PI(−) cell group represents the progress of early apoptosis while the Annexin V(+)/PI(+) cell group underwent later apoptosis. L3-1Y was found to increase both early and late cell apoptosis, compared to 5D5.

4.4. Apoptosis Induction by Anti-c-Met Antibody 2

After L3-1Y or 5D5EBC-1 was applied at various concentrations (0.0032, 0.016, 0.008, 0.4, 2, and 10 μg/ml) to cells which were seeded at a density of 5×104 cells/ml/well into 96-well plates, apoptosis rates were measured in vitro using a Caspase 3/7 Glo® assay system in the same manner as in Example 3.5.

The results are shown in FIG. 4D. As may be seen in FIG. 4D, far higher apoptotic effects on EBC-1 cells were obtained with the antibody L3-1Y (♦) than 5D5 (∘).

4.5. Apoptosis Induction by Anti-c-Met Antibody 3

To examine the relation of LRIG1 to the apoptotic effect, shown in Example 4.4, of the antibody L3-1Y on EBC-1, EBC-1 cells which were subjected to reverse-transfection with LRIG1 siRNAs (Dharmacon) for 24 hours were induced to undergo apoptosis in the same manner as in Example 4.4.

Apoptosis rates are depicted in FIG. 4E. In the graph, sictl represents a control treated with a control siRNA commercially available from Dharmacon. As may be seen in FIG. 4E, the apoptotic effect of L3-1Y (♦) was significantly reduced in LGIG1-knockdown EBC-1 cells compared to the control, indicating that the ability of L3-1Y to induce the apoptosis of EBC-1 cells is attributed to the formation of the LRIG1-c-Met associated protein.

4.6. c-Met Degradation Mechanism of Anti-c-Met Antibody

In order to examine the c-Met degradation mechanism of anti-c-Met antibodies, c-Met degradation was measured in EBC-1 cells whose lysosome pathway was blocked by treatment with the lysosomal pathway inhibitor concanamycin (Merck).

EBC-1 cells which were seeded at a density of 2×105 cells/ml into 96-well plates were incubated at 37° C. for 4 hours with the anti-c-Met antibody L3-1Y 5 μg/ml+DMSO 1 μL, the antibody L3-1Y 5 μg/ml+concanamycin 1 μL, the antibody 5D5 5 μg/ml+DMSO 1 μL, or the antibody 5D5 5 μg/ml+concanamycin 1 μL, and c-Met degradation was measured using ELISA in the same manner as in Example 1.1. For control, IgG 5 μg/ml+DMSO 1 μL, or IgG 5 μg/ml+MG132 1 μL, was used.

The results are given in FIG. 4F. As may be seen in FIG. 4F, c-Met degradation in the 5D5-treated cells was almost equivalent to that in the control cells, and was somewhat increased upon the use of DMSO compared to concanamycin, but without significance because of a difference of only 10% or less. In contrast, the antibody L3-1Y did not trigger c-Met degradation in the presence of concanamycin, but increased c-Met degradation by 60% or greater upon DMSO treatment, demonstrating that the c-Met degradation of the antibody L3-1Y takes the lysosomal pathway.

4.7. Immunocytochemistry

Antibody-mediated co-localization of c-Met and lysosome was analyzed by immunocytochemistry. MKN45 or EBC-1 cells at a density of 2×105 cells/ml were incubated for 4 hours with 1 μg/ml of the anti-c-Met antibody L3-1Y. The cells were fixed with 2% (w/v) paraformaldehyde and blocked for 30 min with 5% (v/v) goat serum (Jackson ImmunoResearch) in PBS-T (0.1% (v/v) Triton X-100 in PBS) before incubation with Alexa 488-conjugated anti-mouse IgG (Invitrogen) at room temperature for 1 hour. Then the cells were washed many times with PBC (Gibco), counterstained with DAPI (4′,6′-diamidine-2′-phenylindole dihydrochloride; Vector Labs) and placed in a fluorescent mounting medium. Immunofluorescence images were obtained with a fluorescence microscope (Carl Zeiss) and are given in FIG. 4G. L3-1Y-APC is an antibody labeled with a detectable tag. As seen in FIG. 4G, the antibody L3-1Y was observed in lysosomes, demonstrating that lysosomes are involved in L3-1Y-triggered c-Met degradation.

Example 5 Therapeutic Effect of Anti-c-Met Antibody on Tumor (In Vivo)

5.1. Anti-Tumor Effect in Tumor-Xenografted Model

For use in examining in vivo effects of anti-c-Met antibody on tumor growth, male BALB/c nude mice, 5˜6 weeks old, were xenografted with a tumor (all experiments were carried out in Pharmalegacy, China). For at least one week before the transplantation, the mice were acclimated.

Then, the mice were anesthetized with 1-2% isofuran and xenografted subcutaneously in the right flank with 5×106 cells of EBC-1 or HS746T. Seven days after the grafting, the tumors measured 50 mm3 or greater on average. The mice were divided into the following three groups: 5D5 (5 mg/kg I.V. once/week) treated, L3-1Y (5 mg/kg I.V. once/week) treated, and vehicle (PBS 0.2 ml I.V. once/week) treated (control). Each group was composed of 15 mice.

Over a total of five weeks, volumes and weights of the tumors were measured 203 times a week. Tumor volume (V) was calculated according to the following formula: V (mm3)={Long Axis Length (mm)×(Small Axis Length (mm))2}/2.

The results are given in FIG. 5C and Table 3 for EBC-1 and in FIG. 5D and Table 4 for HS746T.

TABLE 3 EBC1 At d28:TV Vehicle 0.0 L3-1Y (1) 84.6 L3-1Y (2) 87.0 5D5 23.3

TABLE 4 HS746T At d28:TV Vehicle 0.0 L3-1Y (1) Regression L3-1Y (2) Regression 5D5 56.4 

L3-1Y (1) is an antibody based on hIgG1 with the Th7 hinge (usually referred to just as L3-1Y) while L3-1Y (2) contains the same epitope but has the hIgG2 backbone. FIGS. 5C and 5D are graphs showing tumor growth in mice xenografted with EBC-1 cells (5 c) and HS746T cells (5 d) over time (n=15) (for comparison, the c-Met small molecule inhibitor CPT-11 and a reference antibody (Reference L) were used). Tumor suppression of each of the antibodies was expressed as percentages of volume reduction compared to the tumor volume on the 28th day of the vehicle-treated group (0%) in Table 3 for EBC-1 and Table 4 for HS746T.

As may be seen in FIGS. 5C and 5D and Tables 3 and 4, the antibody L3-1Y was found to have great anti-tumor activity in vivo, compared to the other antibodies.

5.2. Cell Migration Assay

Cell migration was observed in CIM-Plates of the xCELLigence DP system (Roche). A suspension of 5×104 cells in 100 μl of a serum-free medium was placed in the upper chamber. The lower part of CIM-plate 16 was filled with a 10% serum medium (migration to chemo-attractant). In the case that HGF was used, it was added at a concentration of 200 ng/ml to the 10% serum medium. Cell migration capability was evaluated by measuring impedance signals only from the cells that passed through an 8 μm pore membrane. The migration of EBC1 (FIG. 5A) was increased by about 10% upon treatment with 5D5, but decreased by 90% or higher upon treatment with L3-1Y. Only L3-1Y reduced the migration of HS746T, as well, whether treated with HGF or not (FIG. 5B).

Example 6 Anti-Tumor Effect of Anti-c-Met Antibody on Tumor Resistant to EGFR-Targeted Therapy

6.1. Preparation of EGFR-Targeted Therapy-Resistant Tumor

The human lung cancer cell line HCC827 was treated in vitro for a predetermined period of time with Erlotinib, an EGFR antagonist, to establish HCC827 ER (HCC827 Erlotinib-resistant) cell lines. In detail, HCC827 (ATCC) which was seeded at a density of 2×105 cells/ml was treated in vitro with a gradual gradient concentration of the EGFR antagonist Erlotinib (Selleck Chemical) from 5 nm to 10 μm over 5 months to establish Erlotinib-resistant (ER) cell lines HCC827 ER10 and HCC827 ER15.

Erlotinib resistance was confirmed by a cell viability assay, and the results are shown in FIG. 7. The parent cell line HCC827 died with the treatment of Erlotinib or BIBW2992 (Selleck Chemical), whereas the viability of HCC287-ER (Erlotinib resistant) was not reduced at all by Erlotinib. Apart from a high dose of BIBW2992, the ER cells were not induced to undergo cell death by the EGFR antagonist BIBW2992. As may be seen in FIG. 7, the ER cell lines became resistant to the EGFR antagonist BIBW2992 as well as Erlotinib, indicating that there was a likelihood of resistance to other EGFR antagonists.

HCC827, HCC827 ER10, and HCC827 ER15 cells were analyzed for the expression of p-c-Met, c-Met, EGFR, Cbl, LRIG1, and GAPDH therein by immunoblotting using respective antibodies. In this context, antibodies to p-c-Met, c-Met, Cbl, EGFR, and GAPDH (14C10) were purchased from Cell Signaling and an antibody to LRIG1 was purchased from Abcam.

The immunoblots thus obtained are shown in FIG. 6A. As may be seen in FIG. 6A, the level of c-Met gene was about three-fold increased in both the ER cells, compared to non-resistant HCC82, indicating that constant exposure to an EGFR inhibitor increases c-Met levels and thus elicits resistance.

6.2. Apoptotic Effect of a Combination Dosage of Anti-c-Met Antibody and EGFR Antagonist on Tumor Cell

HCC827 ER15, which expresses c-Met at a high level but Cbl at a relatively low level was used in assay for the apoptotic effect of a combination dosage of an anti-c-Met antibody and an EGFR antagonist.

HCC827 ER15 cells were seeded at a density of 5×104 cells/ml/well into 96-well plates and incubated with L3-1Y 0.14 μg/ml, Erlotinib 10 nm, or L3-1Y 0.14 μg/ml+Erlotinib 10 nm at 37° C. for 72 hours. Then, cell viability was measured in the same manner as in Example 1.4.

The results are expressed as percentages of the cell viability (100%) of the control which was neither treated with an anti-c-Met antibody nor an EGFR antagonist in FIG. 6B. As may be seen in FIG. 6B, almost no apoptotic effects were found in the EGFR resistant cells to which the anti-c-Met antibody L3-1Y or the EGFR antagonist Erlotinib were separately administered, but the cell viability was reduced to about 65% when L3-1Y and Erlotinib were administered in combination. That is, a combination dosage of the agents increased the apoptosis of the resistant cells by about 35%.

6.3. Effect of a Combination Dosage of Anti-c-Met Antibody and EGFR Antagonist on c-Met Degradation

An examination was made of the effect of a combination dosage of an anti-c-Met antibody and an EGFR antagonist on c-Met degradation in HCC827 ER15 cells. After HCC827 ER15 cells were treated with a combination of Erlotinib and the antibody L3-1Y or 5D5, levels of c-Met protein in the cells were measured to examine the effect of c-Met antibodies on c-Met degradation.

HCC827 ER15 which was seeded at a density of 2×105 cells/ml/well into 96-well plates was incubated with L3-1Y 0.2 μg/ml+Erlotinib 100 nm, L3-1Y 1.0 μg/ml+Erlotinib 100 nm, 5D5 0.2 μg/ml+Erlotinib 100 nm or 5D5 0.1 μg/ml+Erlotinib 100 nm at 37° C. for 24 hours, and subjected to cell viability assay in the same manner as in Example 1.1, with Erlotinib 100 nm serving as a control.

Cell viability results are expressed as percentages of the c-Met level in the control (treated with Erlotinib alone, 100%) in FIG. 6C, as measured by ELISA. As may be seen in FIG. 6C, a combination of Erlotinib and L3-1Y was found to trigger c-Met degradation in a dose-dependent manner in the Erlotinib-resistant cell line HCC827 ER15, and elicited significant c-Met degradation, compared to a combination of Erlotinib and 5D5.

6.4. Cbl-Independent Inhibitory Activity Against Growth of EGFR-Targeted Therapy-Resistant Sample

For an anticancer effect assay, an experiment was made in which colonies grown from lung cancer patients (LXFE1422, LXFA526, LXFA1647; samples susceptible to c-Met small molecule inhibitors, from Oncotest)-derived cell lines (using a 3D culture medium (RPMI (Gibco)) for non-small cell lung cancer (NSCLC), at 37° C.)) were treated with anti-c-Met antibodies.

Of the lung cancer patients, LXFE1422 was not resistant to Cetuximab, an EGFR-targeting anticancer therapeutic, while both LXFA526 and LXFA1647 patients cannot be treated with Cetuximab due to their resistance.

Lung tissues from the patients were lyzed in a lysis buffer Complete lysis-M (Roche, 04719956001) to separate proteins, followed by Western blot analysis. The results are given in FIG. 6D. As may be seen in FIG. 6D, EGFR and Cbl were present at low levels in LXFA526 and LXFA1647 patients, both resistant to Cetuximab.

From the patient samples, the exon 14 coding for the juxtamembrane domain of c-Met was amplified using a one-step RT-PCR kit with the following primers:

(SEQ ID NO: 106) Primer F: 5′-TGAAATTGAACAGCGAGCTAAAT-3′; and (SEQ ID NO: 107) Primer R: 5′-TTGAAATGCACAATCAGGCTAC-3′.

RT-PCR conditions were as follows:

1 cycle of 50° C. 30 min, 95° C. 15 min,

45 cycles of 94° C. 40 s, 62° C. 40 s, and 72° C. 1 min, and

1 cycle of 72° C. 10 min.

The RT-PCR products including the exon 14 of the c-Met gene were run on agarose gel by electrophoresis, and the agarose gel electrophoresis photograph is given in FIG. 6E. As may be seen, the splice mutation of c-Met itself was found in none of the three patients.

The anticancer activity of the anti-c-Met antibody L3-1Y was proven in the patient samples in which both EGFR and Cbl were expressed at low levels. Cancer cells from the patients were 3D cultured in Oncotest to evaluate colony forming ability. After treatment with L3-1Y 100 μg/ml, the colony forming ability of the cells were evaluated and are expressed as percentages of the number of colonies formed by the control (100 μg/ml treated, 100%) in FIG. 6F. As is apparent from the data of FIG. 6F, the antibody had no efficacy on the LXFE1422 patient, but inhibited cancer growth by 44% (colony formation 56%) for LXFA526, and by 33% (colony formation 67%) for LXFA1647.

Although the preferred embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims.

All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

The use of the terms “a” and “an” and “the” and “at least one” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the term “at least one” followed by a list of one or more items (for example, “at least one of A and B”) is to be construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context. 

What is claimed is:
 1. A method of identifying a subject suitable for treatment with an anti-c-Met antibody or antigen binding fragment thereof that specifically binds to an epitope within a SEMA domain of a c-Met protein, the method comprising: (1) determining a Cbl concentration, a Cbl mutation, and/or a mutation of a site of c-Met for interaction with Cbl in a cell sample from a subject; (2) determining that the subject is suitable for treatment with the anti-c-Met antibody when Cbl is present at a low level or absent in the cell sample or when Cbl or c-Met contains a mutation that inhibits interaction between Cbl and c-Met; and (3) administering the anti-c-Met antibody or antigen-binding fragment thereof to the subject determined to be suitable for treatment, wherein the anti-c-Met antibody or antigen-binding fragment thereof comprises SEQ ID NOs: 1-3, 10, 11, and 13, specifically binds to an epitope having 5 to 19 consecutive amino acids of SEQ ID NO: 71 that includes the amino sequence of SEQ ID NO: 73 (EEPSQ), and promotes LRIG1-mediated c-Met degradation.
 2. The method of claim 1, wherein Cbl concentration is determined by immunohistochemical analysis, and a stain intensity of ‘−’ or ‘+1’ indicates that Cbl is present at a low level or absent, respectively, in the cell sample.
 3. The method of claim 1, wherein the Cbl mutation is a deletion or substitution of 51 or more consecutive nucleotides within nucleotides 1169-1414 of the nucleotide sequence of GenBank Accession Number NM_(—)005188 (SEQ ID NO: 112), or a deletion or substitution of 17 or more consecutive amino acids within amino acids 343-424 of the amino acid sequence of GenBank Accession Number NP_(—)005179 (SEQ ID NO: 113).
 4. The method of claim 1, wherein the mutation of a site of c-Met (SEQ ID NO: 114) for interaction with Cbl is a deletion or a substitution of tyrosine at position 1003 (Y1003) with an amino acid other than tyrosine, or a deletion or a substitution of 141 or more consecutive nucleotides within exon 14 of the c-Met gene with other nucleotides, or a deletion or a substitution of 46 or more consecutive amino acids within a polypeptide encoded by exon 14 of the c-Met gene with other amino acids.
 5. The method of claim 1, further comprising determining the presence of LRIG1 in the cell sample.
 6. The method of claim 5, further comprising determining the cell sample or the subject to be suitable for treatment with the anti-c-Met antibody when LRIG1 is present.
 7. The method of claim 6, wherein LRIG1 concentration is determined by immunohistochemical analysis, and a stain intensity of ‘+2’ or ‘+3’ indicates that LRIG1 is present in the cell sample.
 8. The method of claim 1, wherein the anti-c-Met antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 17 and a light chain variable region comprising an amino acid sequence of SEQ ID NO:
 111. 9. The method of claim 1, wherein the anti-c-Met antibody or antigen binding fragment thereof comprises a heavy chain comprising the amino acid residues from the 18th to 460th positions of SEQ ID NO: 66 and a light chain comprising the amino acid residues from the 21st to 240th positions of SEQ ID NO:
 68. 10. The method of claim 1, wherein the anti-c-Met antibody or antigen binding fragment thereof is an antibody of mouse origin, a mouse-human chimeric antibody, a humanized antibody, or a human antibody.
 11. The method of claim 1, wherein the anti-c-Met antibody or antigen binding fragment thereof is an antibody of mouse origin, a mouse-human chimeric antibody, a humanized antibody, or a human antibody.
 12. The method of claim 1, wherein the cell sample is a sample of cancer cells. 